Modulators of pharmacological agents

ABSTRACT

The biological activity of nucleic acid ligand is regulated (i.e. enhanced or inhibited) in vivo to produce a desired biological effect. This is accomplished through the administration of a modulator, or regulator, that changes the binding of the nucleic acid ligand for its target or that degrades or otherwise cleaves, metabolizes or breaks down the nucleic acid ligand while the ligand is still exerting its effect. Modulators of the present invention can be administered in real time as needed based on various factors, including the progress of the patient, as well as the physician&#39;s discretion in how to achieve optimal therapy. Thus, this invention provides for the first time a regulatable therapeutic regime in the course of nucleic acid ligand therapy.

This application claims priority from U.S. Provisional Application No.60/293,231, filed May 25, 2001, and U.S. Provisional Application No.60/331,037, filed Nov. 7, 2001, the entire contents of both applicationsbeing incorporated herein by reference.

TECHNICAL FIELD

The present invention relates, in general, to an agent that modulatesthe pharmacological activity of a nucleic acid ligand (e.g., aptamer)and, in particular, to an agent that enhances or inhibits the activityof such a ligand. The invention further relates to a compositioncomprising such an agent and to a method of using these agents andcompositions in medical therapeutic and diagnostic procedures.

BACKGROUND

Nucleic acids have conventionally been thought of as primarily playingan informational role in biological processes. Through a method known asSystematic Evolution of Ligands by EXponential enrichment, termed SELEX,it has become clear that nucleic acids have three dimensional structuraldiversity not unlike proteins. SELEX is a method for the in vitrosynthesis and selection of nucleic acid molecules with highly specificbinding to target molecules. The SELEX process was first described byGold and Tuerk in U.S. patent application Ser. No. 07/536,428, filedJun. 11, 1990, now U.S. Pat. No. 5,475,096, and thereafter in U.S.patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled“Methods for Identifying Nucleic Acid Ligands”, now U.S. Pat. No.5,270,163 (see also WO 91/19813). See also Tuerk et al., Science249:505-10 (1990).

Nucleic acid ligands or aptamers are nonencoding single-stranded nucleicacid (DNA or RNA) that have the property of binding specifically to adesired target compound or molecule, and that have sufficient capacityfor forming a variety of two- and three-dimensional structures andsufficient chemical versatility available within their monomers to actas ligands (form specific binding pairs) with virtually any chemicalcompound, whether monomeric or polymeric. Molecules of any size orcomposition can serve as targets. The SELEX method involves selectionfrom a mixture of candidate oligonucleotides and step-wise iterations ofbinding, partitioning and amplification, using the same generalselection scheme, to achieve virtually any desired criterion of bindingaffinity and selectivity. Starting from a mixture of nucleic acids,preferably comprising segments of randomized sequences, the SELEX methodincludes steps of contacting the mixture with the target underconditions favorable for binding, partitioning unbound nucleic acidsfrom those nucleic acids that have bound specifically to targetmolecules, dissociating the nucleic acid-target complexes, amplifyingthe nucleic acids dissociated from the nucleic acid-target complexes toyield a ligand-enriched mixture of nucleic acids, then reiterating thesteps of binding, partitioning, dissociating and amplifying through asmany cycles as desired to yield highly specific high affinity nucleicacid ligands to the target molecule.

Nucleic acid ligands possess a number of features that can render themuseful as therapeutic agents. They can be made as relatively small(e.g., 8 kDa to 15 kDa) synthetic compounds and can be selected topossess high affinity and specificity for target molecules (equilibriumdissociation constants ranging from, for example, 0.05-10 nM). Aptamersembody both the affinity properties of monoclonal antibodies and singlechain antibodies (scFv's) and the manufacturing ease similar to that ofa small peptide. Initial studies demonstrated the in vitro use ofaptamers for studying protein function, and more recent studies haveconfirmed the utility of these compounds for studying in vivo proteinfunction (Floege et al., Am J Pathol 154:169-179 (1999), Ostendorf etal, J Clin Invest 104:913-923, (1999)). In addition, animal studies todate have shown that aptamers and compounds of similar composition arewell tolerated, exhibit low or no immunogenicity, and are thus suitablefor repeated administration as therapeutic compounds (Floege et al., AmJ Pathol 154:169-179 (1999), Ostendorf et al, J Clin Invest 104:913-923(1999), Griffin et al., Blood 81:3271-3276 (1993), Hicke et al., J ClinInvest 106:923-928 (2000)).

As synthetic compounds, site specific modifications (insertions ordeletions) can be made to aptamers to rationally alter theirbioavailability and mode of clearance. For example, it has been foundthat 2′-fluoro pyrimidine-modified aptamers in the 10 kDa to 12 kDa sizerange have a short circulating half-life (˜10 minutes) following bolusintravenous administration but that simple chemical modification of theaptamer or conjugation of the aptamer to a high molecular weight inertcarrier molecule (e.g., PEG) increases circulating half-lifesubstantially (6-12 hours) (Willis et al., Bioconjug Chem 9:573-582(1998), Tucker et al., J Chromatogr Biomed Sci Appl 732:203-212 (1999),Watson et al., Antisense Nucleic Acid Drug Dev 10:63-75 (2000)).Bioactive and nuclease resistant single-stranded nucleic acid ligandscomprising L-nucleotides have been described (Williams et al., Proc.Natl. Acad. Sci. 94:11285 (1997); U.S. Pat. No. 5,780,221; Leva et al.,Chem. Biol. 9:351 (2002)). These “L-aptamers” are reportedly stableunder conditions in which aptamers comprising nucleotides of naturalhandedness (D-nucleotides) (that is, “D-aptamers”) are subject todegradation.

A number of third parties have applied for and secured patents coveringthe identification, manufacture and use of aptamers. As stated above,Larry Gold and Craig Tuerk are generally credited with first developingthe SELEX method for isolating aptamers, and their method is describedin a number of United States patents including those mentioned above andU.S. Pat. Nos. 5,670,637, 5,696,249, 5,843,653, 6,110,900, and5,270,163, as well as other described in the Detailed Description of theInvention. Thomas Bruice et al. reported a process for producingaptamers in U.S. Pat. No. 5,686,242, which differs from the originalSELEX process reported by Tuerk and Gold because it employs strictlyrandom oligonucleotides during the screening sequence. Theoligonucleotides screened in the '242 patent process lack theoligonucleotide primers that are present in oligonucleotides screened inthe SELEX process.

Several patents to Gold et al. relate to aptamers themselves. Forexample, U.S. Pat. No. 6,114,120 relates to an aptamer that binds to acell macromolecule. U.S. Pat. No. 5,670,637 relates to aptamers thatbind to proteins. U.S. Pat. No. 5,696,249 relates to an aptamer producedby the SELEX process.

Other patents have issued that are directed to aptamers against specificbiological targets, and to the methods for identifying these aptamers.U.S. Pat. Nos. 5,756,291 and 5,582,981 to O'Toole, for example, disclosea method for detecting thrombin using a labeled aptamer that comprises adefined six nucleotide sequence. U.S. Pat. Nos. 5,527,894 and 5,637,461of Gold et al. relate to methods of identifying aptamers against the tatprotein. Other patents that disclose aptamers directed against specificbiological targets include U.S. Pat. Nos. 5,496,938 (HIV-reversetranscriptase), 5,476,766 (thrombin), 5,459,015 (fibroblast growthfactor), 5,472,841 (neutrophil elastase), 5,849,479 (vascularendothelial growth factor), 5,726,017 (HIV GAG), 5,731,144 (TGFβ),5,827,456 (chorionic gonadotropin hormone), 5,780,228 (lectins),5,766,853 (selectins), 5,674,685 (platelet derived growth factor),5,763,173 (DNA polymerases), 6,140,490. (complement system proteins),and 5,869,641 (CD4).

Sullenger, Rusconi, Kontos and White in WO 0226932 A2 describe RNAaptamers that bind to coagulation factors, E2F family transcriptionfactors, Ang1, Ang2, and fragments or peptides thereof, transcriptionfactors, autoimmune antibodies and cell surface receptors useful in themodulation of hemostasis and other biologic events. (See also Rusconi etal., Thrombosis and Haemostasis 83:841-848 (2000), White et al., J. ClinInvest 106:929-34 (2000), Ishizaki et al., Nat Med 2:1386-1389 (1996),and Lee et al., Nat Biotechnol 15:41-45 (1997)).

A number of patents have also issued that relate to specific uses ofaptamers. For example, Bruice et al. in U.S. Pat. No. 6,022,691 describethe use of aptamers identified by a SELEX-like process to detect drugsand other molecules in biological fluids. Gold et al. in U.S. Pat. No.5,843,653 provide a diagnostic method using aptamers. U.S. Pat. No.6,110,900 discloses a diagnostic composition that contains an aptamer.U.S. Pat. No. 5,789,163 discloses a sandwich assay that employs aptamersas the capture and/or detection ligand. U.S. Pat. No. 6,147,204describes the use of aptamers/lipophile complexes to deliver therapeuticand diagnostic aptamers to intracellular locations in vivo. U.S. Pat.Nos. 5,705,337, 5,962,219, 5,763,595 and 5,998,142 disclose aptamersthat are chemically modified to covalently bind to target proteins.

Several methods have been developed that modify the base SELEX processto obtain aptamers that satisfy objectives in addition to exhibitinghigh binding affinity toward a target molecule. For example, a number ofpatents disclose the use of modified nucleotides in the SELEX process toobtain aptamers that exhibit improved properties. U.S. Pat. No.5,660,985, for example, relates to SELEX using 2′-modified nucleotidesthat display enhanced in vivo stability. U.S. Pat. No. 6,083,696discloses a “blended” SELEX process in which oligonucleotides covalentlylinked to non-nucleic acid functional units are screened for theircapacity to bind a target molecule. Other patents describe post-SELEXmodifications to aptamers to decrease their size, increase theirstability, or increase target binding affinity. See, e.g., U.S. Pat.Nos. 5,817,785 and 5,648,214.

Still other patents describe unique SELEX processes. For example, U.S.Pat. Nos. 5,763,566 and 6,114,120 disclose processes for generatingaptamers using the SELEX process with whole biological tissue as thetarget, to identify aptamers that have binding affinity towardbiological tissues and components thereof. U.S. Pat. No. 5,580,737discloses a modification to the SELEX process that yields aptamers thatcan discriminate between two or more compounds. U.S. Pat. No. 5,567,588discloses the “solution SELEX” method in which the nucleic acidcandidate mixture is screened in solution in order to preferentiallyamplify the highest affinity aptamer.

Kauffman has obtained patents disclosing the generation of largelibraries of proteins from large pools of stochastically generatedoligonucleotide vectors. See U.S. Pat. Nos. 5,814,476 and 5,723,323.

Weis et al. disclose in U.S. Pat. No. 5,245,022 an oligonucleotide ofabout 12-25 bases that is terminally substituted by apolyalkyleneglycol. These modified oligonucleotides are reported to beresistant to exonuclease activity.

U.S. Pat. Nos. 5,670,633 and 6,005,087 to Cook et al. describe thermallystable 2′-fluoro oligonucleotides that are complementary to an RNA orDNA base sequence. U.S. Pat. Nos. 6,222,025 and 5,760,202 to Cook et al.describe the synthesis of 2′-O substituted pyrimidines and oligomerscontaining the modified pyrimidines. EP 0 593 901 B1 disclosesoligonucleotide and ribozyme analogues with terminal 3′,3′- and5′,5′-nucleoside bonds. U.S. Pat. No. 6,011,020 to Gold et al. disclosesan aptamer modified by polyethylene glycol.

A number of U.S. patents have issued that describe methods of largescale manufacturing that can be used to produce aptamers. Caruthers etal., for example, describe in U.S. Pat. Nos. 4,973,679; 4,668,777; and4,415,732 a class of phosphoramidite compounds that are useful in themanufacture of oligonucleotides. In another series of patents, Carutherset al. disclose a method of synthesizing oligonucleotides using aninorganic polymer support. See, e.g., U.S. Pat. Nos. 4,500,707,4,458,066 and 5,153,319. In still another series of patents, Carutherset al. discloses a class of nucleoside phosphorodithioates that can beused to manufacture oligonucleotides. See, e.g., U.S. Pat. Nos.5,278,302, 5,453,496 and 5,602,244. Reports of aptamers designed to bindto other aptamers include: Aldaz-Carroll L, Tallet B, Dausse E,Yurchenko L, Toulme J J.; Apical loop-internal loop interactions: a newRNA-RNA recognition motif identified through in vitro selection againstRNA hairpins of the hepatitis C virus mRNA; Biochemistry. 2002 May 7;41(18):5883-93; Darfeuille F, Cazenave C, Gryaznov S, Duconge F, DiPrimo C, Toulme J J.; RNA and N3′->P5′ kissing aptamers targeted to thetrans-activation responsive (TAR) RNA of the human immunodeficiencyvirus-1, Nucleosides Nucleotides Nucleic Acids. 2001Apr.-Jul.;20(4-7):441-9; Collin D, van Heijenoort C, Boiziau C, Toulme JJ, Guittet E., NMR characterization of a kissing complex formed betweenthe TAR RNA element of HIV-1 and a DNA aptamer. Nucleic Acids Res. 2000Sep. 1; 28(17):3386-91; Duconge F, Di Primo C, Toulme J J., Is a closing“GA pair” a rule for stable loop-loop RNA complexes? J Biol. Chem. 2000Jul. 14; 275(28):21287-94.; Duconge F, Toulme J J. In vitro selectionidentifies key determinants for loop-loop interactions: RNA aptamersselective for the TAR RNA element of HIV-1. RNA. 1999 December; 5(12):1605-14; Boiziau C, Dausse E, Yurchenko L, Toulme J J., DNA aptamersselected against the HIV-1 trans-activation-responsive RNA element formRNA-DNA kissing complexes; J Biol. Chem. 1999 Apr. 30; 274(18):12730-7;and Le Tinevez R, Mishra R K, Toulme J J., Selective inhibition ofcell-free translation by oligonucleotides targeted to a mRNA hairpinstructure; Nucleic Acids Res. 1998 May 15; 26(10):2273-8.

Currently, many drugs elicit medical complications such as side effectsand undesirable or uncontrollable outcomes. Treating medicalcomplications that result from side effects leads to additionalhealthcare costs. The recent identification of this range of nucleicacid ligands useful in medical therapy has opened new avenues ofresearch and development. While progress has been made in this area, astrong need remains to provide methods and compositions to improve themanner in which these ligands are used and to increase their efficacy,to better control the process of therapy, and to provide therapies thathave decreased side effects over traditional therapeutic methods. Thepresent invention provides compounds, compositions and methods toimprove the process of using nucleic acid ligands in medical therapy.The approach provided by the present invention allows for more controlover the therapeutic effect, pharmacokinetics and duration of activityof nucleic acid ligands.

SUMMARY OF THE INVENTION

It has been discovered that the biological activity of nucleic acidligands can be modulated (i.e., enhanced or inhibited) in vivo toproduce a desired biological effect. This can be accomplished throughthe administration of a modulator, or regulator, that changes thebinding of the nucleic acid ligand for its target or that degrades orotherwise cleaves, metabolizes or breaks down the nucleic acid ligandwhile the ligand is still exerting its effect. Modulators of the presentinvention can be administered in real time as needed based on variousfactors, including the progress of the patient, as well as thephysician's discretion in how to achieve optimal therapy. Thus, thisinvention provides for the first time a regulatable therapeutic regimein the course of nucleic acid ligand therapy.

This regulatable therapeutic regime controls drug action by introducinga modulator that is easy to use, can be independent of the patient'shealth status, has a uniform mode of action, and does not requirecontinuous drug infusion. In one example, an antidote is provided thatis rationally designed to turn off aptamer activity when desired by thephysician.

The modulator can be a oligonucleotide, a small molecule, a peptide,oligosaccharide, for example an aminoglycoside, or other molecule thatcan bind to or otherwise modulate the activity of the therapeuticnucleic acid ligand, including a small molecule, or a chimera or fusionor linked product of any of these. For example, the modulator can be anoligonucleotide that is complementary to at least a portion of thenucleic acid ligand. In another embodiment, the modulator can be aribozyme or DNAzyme that targets the nucleic acid ligand. In a furtherembodiment, the modulator can be a peptide nucleic acid (PNA),morpholino nucleic acid (MNA), locked nucleic acid (LNA) or pseudocyclicoligonucleobases (PCO) that includes a sequence that is complementary toor hybridizes with at least a portion of the nucleic acid ligand. Atypical nucleic acid ligand (e.g., aptamer) possesses some amount ofsecondary structure—its active tertiary structure is dependent onformation of the appropriate stable secondary structure. Therefore,while the mechanism of formation of a duplex between a complementaryoligonucleotide modulator of the invention and a nucleic acid ligand isthe same as between two short linear oligoribonucleotides, both therules for designing such interactions and the kinetics of formation ofsuch a product are impacted by the intramolecular aptamer structure. Therate of nucleation is important for formation of the final stableduplex, and the rate of this step is greatly enhanced by targeting theoligonucleotide modulator to single-stranded loops and/orsingle-stranded 3′ or 5′ tails present in the nucleic acid ligand. Forthe formation of the intermolecular duplex to occur, the free energy offormation of the intermolecular duplex has to be favorable with respectto formation of the existing intramolecular duplexes within the targetednucleic acid ligand.

In an alternative embodiment of the invention, the modulator itself isan aptamer. In this embodiment, a nucleic acid ligand is first generatedthat binds to the desired therapeutic target. In a second step, a secondnucleic acid ligand that binds to the first nucleic acid ligand isgenerated using the SELEX process described herein or other process, andmodulates the interaction between the therapeutic nucleic acid ligandand the target. In one embodiment, the second nucleic acid liganddeactivates the effect of the first nucleic acid ligand.

In other alternative embodiments, the aptamer which binds to the targetcan be a PNA, MNA, LNA or PCO and the modulator is a nucleic acidligand. Alternatively, the aptamer which binds to the target is a PNA,MNA, LNA or PCO, and the modulator is a PNA. Alternatively, the aptamerwhich binds to the target is a PNA, MNA, LNA or PCO, and the modulatoris an MNA. Alternatively, the aptamer which binds to the target is aPNA, MNA, LNA or PCO, and the modulator is an LNA. Alternatively, theaptamer which binds to the target is a PNA, MNA, LNA or PCO, and themodulator is a PCO. Any of these can be used, as desired, in thenaturally occurring stereochemistry or in non-naturally occurringstereochemistry or a mixture thereof. For example, in a preferredembodiment, the nucleic acid ligand is in the D configuration, and in analternative embodiment, the nucleic acid ligand is in the Lconfiguration.

The present invention also provides methods to identify the modulatorsof nucleic acid ligands. Modulators can be identified in general,through binding assays, molecular modeling, or in vivo or in vitroassays that measure the modification of biological function. In oneembodiment, the binding of a modulator to a nucleic acid is determinedby a gel shift assay. In another embodiment, the binding of a modulatorto a nucleic acid ligand is determined by a Biacore assay. Otherappropriate assays are described in the Detailed Description of theInvention.

In another embodiment, the binding or interaction of the modulator withthe nucleic acid ligand is measured by evaluating the effect of thenucleic acid ligand with and without the modulator under appropriatebiological conditions. As an example, modulators of the invention can beidentified which regulate antithrombotic and anticoagulant aptamers.Modulator efficacy can be assessed in vitro or in vivo through acoagulation test bioassay such as the activated coagulation time test,the activated partial thromboplastin test, the bleeding time test, theprothrombin time test, or the thrombin clotting time test. Using anidentified regime, a patient can be administered an anticoagulantnucleic acid ligand and then given the antidote when the time isappropriate to resume normal clotting action. This regime is useful, forexample, during cardiovascular and vascular surgery, percutaneouscoronary interventions (angioplasty), orthopedic surgery, and treatmentof acute myocardial infarction. In a non-limiting, illustrative example,modulators of the present invention can bind to nucleic acid ligandsthat target tissue factor (TF)/factor VIa (FVIIa), factor VIIIa(FVIIIa)/factor IXa (FIXa), factor Va (FVa/factor Xa (Fxa) enzymecomplexes and platelet receptors such as gp IIbIIIa and gp IbIX andmodulate the effects of the nucleic acid ligand. This invention alsoprovides antidote controlled platelet inhibitors, antithrombotics andfibrinolytics.

In a one embodiment, the modulator is an oligonucleotide that binds to aFactor IXa aptamer (for example, Aptamer 9.3 or Aptamer 9.3t) thattargets Coagulation Factor IXa. The antidote oligonucleotide can becomplementary to at least a portion of the Factor IXa aptamer.Specifically, the antidote 2′-O-methyl oligonucleotide can consist ofthe following sequence, 5′AUGGGGAGGCAGCAUUA 3′, 5′CAUGGGGAGGCAGCAUUA3′,5′CAUGGGGAGGCAGCA3′, 5′CAUGGGGAGGCA3′, 5′GCAUUACGCGGUAUAGUCCCCUA3′,5′CGCGGUAUAGUCCCCUA3′, 5′CGC GGU AUA GUC CCC AU3′. Modifications orderivatives thereof wherein a desired degree of hybridization ismaintained.

In another embodiment, the modulator is an oligonucleotide that binds toa Factor Xa aptamer (for example, Aptamer 11F7t) that targetsCoagulation Factor Xa. The antidote oligonucleotide can be complementaryto at least a portion of the Factor Xa aptamer. Specifically, theantidote oligonucleotide may consist of the following sequences,5′CUCGCUGGGGCUCUC3′, 5′UAUUAUCUCGCUGGG3′, 5′AAGAGCGGGGCCAAG3′,5′GGGCCAAGUAUUAU 3′, 5′CAAGAGCGGGGCCAAG 3′, 5′CGAGUAUUAUCUUG3′ or anymodification or derivative thereof wherein a desired degree ofhybridization is maintained.

In another embodiment, the oligonucleotide modulators include nucleicacid sequences that are substantially homologous to and that havesubstantially the same ability to bind nucleic acid ligands, as theoligonucleotide modulators identified herein.

In a further embodiment, modulators of the invention can also be used toreverse the immunosuppressive effect of nucleic acid ligands that targetinterleukin, for example, in patients subject to infection. The presentmodulators can be used to reverse the immunostimulatory effects ofnucleic acid ligands that target CTLA4 in patients at risk of developingautoimmunity.

In a further embodiment, modulators of the invention can be used toreverse the effects of aptamers that target growth factors (e.g., PDGFor VEGF). Such nucleic acid ligands can be used in the treatment oftumors and in the treatment of inflammatory proliferative diseases.Since growth factors play systemic roles in normal cell survival andproliferation, nucleic acid ligand treatment can result in a breakdownof healthy tissue if not tightly regulated (e.g., patients receivingnucleic acid ligands that target angiopoietin I can be subject tohemorrhaging). Modulators of the invention that target such nucleic acidligands can be used to provide the necessary regulation.

Modulators of the invention can be used to reverse the effects ofnucleic acid ligands that target receptors involved in the transmissionof the nerve impulse at the neuromuscular junction of skeletal muscleand/or autonomic ganglia (e.g., nicotinic acetylcholine or nicotiniccholinergic receptors). Such nucleic acid ligands can be made to producemuscular relaxation or paralysis during anesthesia. Agents that blockthe activity of acetylcholine receptors (agents that engenderneuromuscular blockade) are commonly used during surgical procedures,and it is preferred that the patients regain muscular function as soonas possible after the surgical procedure is complete to reducecomplications and improve patient turnover in the operating arenas.Therefore, much effort has been made to generate agents with predictablepharmacokinetics to match the duration of the drug activity to theanticipated duration of the surgical procedure. Alternatively,modulators of the invention that target such nucleic acid ligands can beused to provide the desired control of the activity of the neuromuscularblocker, and thus reduce the dependence on the patient's physiology toprovide reversal of the neuromuscular blocking agent.

In a still further embodiment, modulators of the invention can be usedto reverse the effect of nucleic acid ligands that target smallmolecules, such as glucose. Hypoglycemia can be avoided in patientsreceiving glucose-targeted nucleic acid ligands to regulate glucoseuptake using the modulators of the invention.

Further, modulators can also be used to regulate the activity of nucleicacid ligands directed against members of the E2F family, certain ofwhich are pro-proliferative, certain of which are repressive. Themodulators of the invention can be used to “turn on” and “turn off” suchnucleic acid ligands at desired points in the cell cycle.

In another embodiment, modulators of the invention can also be used toreverse the binding of nucleic acid ligands bearing radioactive orcytotoxic moieties to target tissue (e.g., neoplastic tissue) andthereby, for example, facilitate clearance of such moieties from apatient's system. Similarly, the modulators of the invention can be usedto reverse the binding of nucleic acid ligands labeled with detectablemoieties (used, for example, in imaging or cell isolation or sorting) totarget cells or tissues (Hicke et al. J. Clin. Invest. 106:923 (2000);Ringquist et al., Cytometry 33:394 (1998)). This reversal can be used toexpedite clearance of the detectable moiety from a patient's system.

Modulators of the invention can also be used in in vitro settings toenhance or inhibit the effect of a nucleic acid ligand (e.g., aptamer)on a target molecule. For example, modulators of the invention can beused in target validation studies. Using modulators of the invention, itis possible to confirm that a response observed after inhibiting atarget molecule (with a nucleic acid ligand) is due to specificallyinhibiting that molecule.

The modulators of the invention can be formulated into pharmaceuticalcompositions that can include, in addition to the modulator, apharmaceutically acceptable carrier, diluent or excipient. The precisenature of the composition will depend, at least in part, on the natureof the modulator and the route of administration. Optimum dosingregimens can be readily established by one skilled in the art and canvary with the modulator, the patient and the effect sought. Generally,the modulator can be administered IV, IM, IP, SC, or topically, asappropriate.

Alternatively, and in view of the specificity of the modulators of theinvention, subsequent treatment can involve the administration ofadditional nucleic acid ligands that are either the same or different asthe original nucleic acid ligand/antidote oligonucleotide pair firstadministered.

Finally, one optional embodiment of the invention is the identificationand selection of modulators and regulators that exhibit relevant speciescross-reactivity to increase usefulness of preclinical animal studies.

Objects and advantages of the present invention will be clear from thedescription that follows:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the FIXa aptamer 9.3t (SEQ ID NO: 1). All purines are2′-hydroxyl and all pyrimidines are 2′-fluoro nucleotides.

FIG. 2 shows the anticoagulant activity of aptamer 9.3t. Clot timeincrease is normalized to baseline clot time in the absence of aptamer.

FIG. 3 shows ACT change of pigs following aptamer injection. Data arenormalized to pre-injection baseline.

FIG. 4 shows the clot time change of pigs following aptamer injection.

FIGS. 5A-5C show the in vitro inhibitory activity ofcholesterol-modified aptamer, 9.3t-C. FIG. 5A. Cholesterol addition hasa modest effect on the affinity of aptamer 9.3t-C for FIXa. Acompetition binding assay is used to measure affinity of 9.3t-C forFIXa. FIG. 5B. In vitro anti-coagulant activity of aptamer 9.3t-C inhuman plasma. FIG. 5C. In vitro anticoagulant activity of aptamer 9.3t-Cin pig plasma.

FIGS. 6A-6C show the in vivo anticoagulant activity of aptamer 9.3t-C.FIG. 6A. In vivo anticoagulant activity of aptamer 9.3t-C in pigsfollowing IV bolus injection, ACT assays (dotted line is 9.3t ACT dataat 0.5 mg/ml from FIG. 3). FIG. 6B. In vivo anticoagulant activity ofaptamer 9.3t-C in pigs following IV bolus injection, APTT and PT assays(0.5 mg/kg 9.3t from FIG. 4). FIG. 6C. In vivo plasma concentration of9.3t-C versus 9.3t over time following bolus IV injection.Concentrations were calculated by interpolation from in vitro doseresponse curves of APTT assays for each aptamer.

FIG. 7 shows alignment of minimal FIXa aptamers (SEQ ID NO:2-SEQ ID NO:17). Sequences in lower case letters are derived from the fixed regionof the library used in the SELEX process and sequences in upper caseletters are derived from the random region of the library used in theSELEX process. S=stem, L=loop.

FIG. 8 shows the native gel analysis of binding of antidoteoligonucleotide to aptamer 9.3t. The ability of the antidoteoligonucleotide to bind to and denature aptamer 9.3t was evaluated bynative gel electrophoresis. Briefly, radiolabeled 9.3t (125 nM) wasincubated at 37° C. for 15 minutes with (from left to right) an 8 fold,4 fold, 2 fold molar excess or equimolar amounts of the antidote (A.S.)or nonsense oligonucleotide (N.S.). Native 9.3t migrates faster thanantidote bound 9.3t in this gel system (compare lanes 1 and 2).

FIGS. 9A and 9B show antidote oligonucleotide reversal of anticoagulantactivity of aptamer 9.3t in human plasma. FIG. 9A. Change in clot timeversus antidote or nonsense oligonucleotide concentration. A value of1.0 indicates no change in clot time over the baseline value. 9.3tM is amutant version of aptamer 9.3t that has no anticoagulant activity. FIG.9B. Fraction of the anticoagulant activity of aptamer 9.3t reversedversus the molar excess of antidote oligonucleotide.

FIG. 10A-10C show the specificity of oligonucleotide antidotes. FIG.10A. Minimal secondary structure of aptamer 9.20t (SEQ ID NO: 18). FIG.10B Anticoagulant activity of 9.20t. FIG. 10C. Specificity of antidoteoligonucleotide Anti D1.

FIG. 11 shows the secondary structure of tailed aptamer 9.3t-3NT (SEQ IDNO: 19). Also shown are antidote oligonucleotides (SEQ ID NO:20-SEQ IDNO:22).

FIGS. 12A and 12B show the activity of aptamer 9.3t-3NT. FIG. 12A.Competitive binding data. FIG. 12B. In vitro anticoagulant data.

FIGS. 13A and 13B show the in vitro reversal of the anticoagulantactivity of aptamer 9.3t-3NT. FIG. 13A. Reversal of anticoagulantactivity vs. concentration of antidote oligonucleotide. FIG. 13B.Reversal of anticoagulant activity vs. molar excess of antidote overaptamer.

FIGS. 14A and 14B show the impact of tail addition to FIXa aptamer 9.3ton the ability to reverse anticoagulant activity.

FIG. 15. The antidote oligonucleotide 5-2C but not a scrambled versionof this antidote oligonucleotide, 5-2C scr, effectively reverses theactivity of aptamers 9.3t and Peg-9.3t in human plasma.

FIG. 16. Kinetics of antidote activity in human plasma, as described inExample 6.

FIG. 17. Duration of antidote activity in vitro, as described in Example6.

FIGS. 18A and 18B. FIG. 18A. Predicted secondary structure of aptamer11F7t (SEQ ID NO:23), which binds human coagulation factor Xa with aK_(D) of ˜1.5 nM. FIG. 18B. Predicted secondary structure of a mutantversion of aptamer 11F7t, termed 11F7tM (SEQ ID NO:24).

FIGS. 19A and 19B. Aptamer 11F7t is a potent anticoagulant of humanplasma. Varying concentrations of aptamer 11F7t were added to humanplasma in vitro, and the clot time was then measured in a PT (FIG. 19A)or APTT assay (FIG. 19B). All data are normalized to the baseline forthat day, so that a value of 1 equals no change in clot time.

FIG. 20. Sequences of 11F7t to which the antidote oligos arecomplimentary.

FIGS. 21A and 21B. FIG. 21A. The antidote oligonucleotides effectivelyreverse the activity of aptamer 11F7t in human plasma. FIG. 21B.Characterization of antidote 5-2 activity over a larger concentrationrange of antidote 5-2, and comparison to the antidote activity of ascrambled sequence version of antidote 5-2, 5-2 scr.

FIG. 22. Kinetics of antidote activity in human plasma. Aptamer 11F7twas added to human plasma in vitro at a final concentration of 125 nM,and allowed to incubate for 5 minutes at 37° C. Antidote oligonucleotide5-2 at a 1:1 or 5:1 molar excess or no antidote were then added, and theclotting activity of the plasma determined by measuring the clottingtime in a PT assay at the times indicated following antidote addition.The % residual anticoagulant activity remaining was calculated bycomparing the difference over baseline between the clotting time in thepresence of antidote to the difference over baseline in the absence ofantidote at each time point. The data collected at a 5:1 molar excess ofantidote 5-2 to aptamer 11F7t is not shown, as reversal of theanticoagulant activity was complete at the first time point (1 minute).

FIG. 23. Duration of antidote activity in vitro. The duration of theinactivation of the anticoagulant activity of aptamer 11F7t by antidoteoligonucleotide 5-2 was measured in vitro in human plasma. Briefly,11F7t was added to human plasma to a final concentration of 125 mM andallowed to incubate for 5 minutes. Antidote oligonucleotide 5-2 was thenadded at a 4 fold molar excess, or in a parallel experiment buffer alonewas added in place of the antidote oligo, and the clotting time wasmeasured in an a PT assay at various time points following antidoteaddition. All data is normalized to the baseline for that day, so that avalue of 1=no change in clot time. It was found that after 5 hours ofincubation at 37° C., the PT of the untreated plasma began to increase,indicating the loss of the clot forming activity of the plasma, and theexperiment was thus stopped at 5 hours.

FIG. 24. Aptamers 9.3t and 11F7t and their respective antidotes functionindependent of each other in human plasma. Apt1=9.3t (30 nM), Apt2=11F7t(100 nM), AD1=AO 5−2c (300 nM), AD2=AO5=2 (500 nM)-final concentrationsin plasma in ( ). Aptamers were added to human plasma at 37° C. asindicated, and allowed to incubate for 5 minutes. Antidotes were thenadded as indicated, and the clotting activity was measured 10 minutesafter antidote addition in APTT assays. In all assays, buffer alone wassubstituted for aptamer or antidote in cases in which only one aptameror antidote was added to the plasma. All data is normalized to thebaseline for that day, so that a value of 1=no change in clot time.

FIGS. 25A-25F. Antidote-controlled anticoagulation of plasma frompatients with heparin-induced thrombocytopenia. FIGS. 25A-25C. Theactivity of aptamer Peg-9.3t and antidote 5-2 were tested in plasma fromhemodialysis-dependent patients diagnosed with HIT. FIG. 25D-25F. Theactivity of aptamer Peg-9.3t and antidote 5-2 were tested in plasma frompatients suffering from thromboembolic complications of HIT. Plasmasamples were treated as indicated: aptamer, 125 nM Peg-9.3t; antidote,1.25 μM AO 5-2; mutant aptamer, 125 nM 9.3tM. Experiments were performedas described in Example 2, FIG. 9. Data is reported in seconds (s) andis the average ± range of duplicate measurements.

FIGS. 26A and B. Antidote-controlled anticoagulation of plasma frompatients with heparin-induced thrombocytopenia. The activity of aptamer11F7t and antidote 5-2 were tested in plasma from ahemodialysis-dependent patient diagnosed with HIT and from a patientsuffering from thromboembolic complications of HIT. For patient 3,plasma samples were treated as indicated: aptamer, 250 nM 11F7t;antidote, 1.0 μM AO 5-2; mutant aptamer, 250 nM 9.3tM. Experiments wereperformed as described in Example 2, FIG. 9. For Patient 6, plasmasamples were treated as indicated: aptamer, 125 nM 11F7t; antidote, 250nM AO 5-2; mutant aptamer, 125 nM 9.3tM. Data is reported in seconds (s)and is the average ± range of duplicate measurements.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to modulators of pharmacologicalagents, including therapeutic and diagnostic agents. The inventionfurther relates to methods of enhancing or inhibiting the efficacy ofpharmacological agents by administering modulators of the invention to asubject (e.g., a human) in need thereof. Additionally, the inventionrelates to methods of using modulators of the invention to assess theactivity of nucleic acid ligands, in vivo and in vitro.

The present invention relates to a method of modulating the activity ofa nucleic acid ligand, for example, by altering its conformation andthus its function. In accordance with the invention, the modulator canbe contacted with the targeted nucleic acid ligand under conditions suchthat it binds to the nucleic acid ligand and modifies the interactionbetween the nucleic acid ligand and its target molecule. Modification ofthat interaction can result from modification of the nucleic acid ligandstructure as a result of binding by the modulator. The modulator canbind the free nucleic acid ligand and/or the nucleic acid ligand boundto its target molecule.

Modulators of the invention can be designed so as to bind any particularnucleic acid ligand with a high degree of specificity and a desireddegree of affinity. Modulators can be also be designed so that, uponbinding, the structure of the nucleic acid ligand is modified to eithera more or less active form. For example, the modulator can be designedso that upon binding to the targeted nucleic acid ligand, thethree-dimensional structure of that nucleic acid ligand is altered suchthat the nucleic acid ligand can no longer bind to its target moleculeor binds to its target molecule with less affinity.

Alternatively, the modulator can be designed so that, upon binding, thethree dimensional structure of the nucleic acid ligand is altered sothat the affinity of the nucleic acid ligand for its target molecule isenhanced. That is, the modulator can be designed so that, upon binding,a structural motif is produced in the nucleic acid ligand so that thenucleic acid ligand can bind to its target molecule.

In one embodiment, the modulator is an oligonucleotide. Theoligonucleotide can be a sequence that is complementary to at least aportion of the nucleic acid ligand. In another embodiment, the modulatoris a ribozyme or DNAzyme that targets the nucleic acid ligand. In afurther embodiment, the modulator can be, for example, a peptide nucleicacid or morpholino nucleic acid that includes a sequence that iscomplementary to or hybridizes with at least a portion of the nucleicacid ligand. A modulator is specifically hybridizable with the nucleicacid ligand when binding of the modulator to the nucleic acid ligandsufficiently interferes with the normal function of the nucleic acidligand to cause a change in the biological activity of the nucleic acidligand, under physiological conditions. In an alternative embodiment,there is a sufficient degree of non-Watson Crick binding of themodulator to the nucleic acid ligand to affect the activity of thenucleic acid ligand.

In a still further embodiment, the modulator is nucleic acid bindingpeptide, polypeptide or protein that binds to or otherwise interactswith the nucleic acid ligand. In a further embodiment, the modulator isan oligosaccharide that binds the nucleic acid ligand. In a specificembodiment, the modulator is an aminoglycoside. In another embodiment,the modulator is a small organic molecule (i.e., a molecule that can besynthetic or naturally occurring that is not otherwise found in vivo andtypically has a molecular weight of less than 1000).

The present invention also includes methods for identifying modulatorsof nucleic acid ligands. In one embodiment, the binding of a modulatorto a nucleic acid is determined by any assay that measures bindingaffinity, such as a gel shift assay. In another embodiment, the bindingof a modulator to a nucleic acid ligand is determined by a Biacoreassay. Other exemplary assays are described below.

In another embodiment, the binding or interaction of the modulator withthe nucleic acid ligand is measured by evaluating the effect of theligand with and without the regulator under appropriate biologicalconditions. For example, modulators can be identified that modify theantithrombotic or anticoagulant activity of nucleic acid ligand in vitroor in vivo through a coagulation test bioassay such as an activatedcoagulation time test, the activated partial thromboplastin test, thebleeding time test, the prothrombin time test, or the thrombin clottingtime test.

The present invention further includes the use of such modulators in avariety of indications whereby control of nucleic acid ligand activityis desired. The modulators may act to inhibit nucleic acid ligandactivity as an antidote to reverse the actions of the nucleic acidligand. Additionally, the invention provides methods of using modulatorsof the invention to assess the activity of nucleic acid ligands. Theinvention is also directed to methods of enhancing or inhibiting theefficacy of nucleic acid ligands by administering modulators of theinvention to human or non-human mammals.

In a further embodiment, modulators of the invention can also be used toreverse the immunosuppressive effect of nucleic acid ligands that targetinterleukin, for example, in patients subject to infection. The presentmodulators can be used to reverse the immunostimulatory effects ofnucleic acid ligands that target CTLA4 in patients at risk of developingautoimmunity.

In a further embodiment, modulators of the invention can be used toreverse the effects of nucleic acid ligands that target growth factors(e.g., PDGF or VEGF). Such nucleic acid ligands can be used in thetreatment of tumors and in the treatment of inflammatory proliferativediseases. Since growth factors play systemic roles in normal cellsurvival and proliferation, nucleic acid ligand treatment can result ina breakdown of healthy tissue if not tightly regulated (e.g., patientsreceiving nucleic acid ligands that target angiopoietin I can be subjectto hemorrhaging). Modulators of the invention that target such nucleicacid ligands can be used to provide the necessary regulation.

Modulators of the invention can be used to reverse the effects ofnucleic acid ligands that target receptors involved in the transmissionof the nerve impulse at the neuromuscular junction of skeletal muscleand/or autonomic ganglia (e.g., nicotinic acetylcholine or nicotiniccholinergic receptors). Such nucleic acid ligands can be made to producemuscular relaxation or paralysis during anesthesia. Agents that blockthe activity of acetylcholine receptors (agents that engenderneuromuscular blockade) are commonly used during surgical procedures,and it is preferred that the patients regain muscular function as soonas possible after the surgical procedure is complete to reducecomplications and improve patient turnover in the operating arenas.Therefore, much effort has been made to generate agents with predictablepharmacokinetics to match the duration of the drug activity to theanticipated duration of the surgical procedure. Alternatively,modulators of the invention that target such nucleic acid ligands can beused to provide the desired control of the activity of the neuromuscularblocker, and thus reduce the dependence on the patient's physiology toprovide reversal of the neuromuscular blocking agent.

In a still further embodiment, modulators of the invention can be usedto reverse the effect of nucleic acid ligands that target smallmolecules, such as glucose. Hypoglycemia can be avoided in patientsreceiving glucose-targeted nucleic acid ligands to regulate glucoseuptake using the modulators of the invention.

Further, modulators can also be used to regulate the activity of nucleicacid ligands directed against members of the E2F family, certain ofwhich are pro-proliferative, certain of which are repressive. Themodulators of the invention can be used to “turn on” and “turn off” suchnucleic acid ligands at desired points in the cell cycle.

In another embodiment, modulators of the invention can also be used toreverse the binding of nucleic acid ligands bearing radioactive orcytotoxic moities to target tissue (e.g., neoplastic tissue) andthereby, for example, facilitate clearance of such moeity's from apatient's system. Similarly, the modulators of the invention can be usedto reverse the binding of nucleic acid ligands labeled with detectablemoieties (used, for example, in imaging or cell isolation or sorting) totarget cells or tissues (Hicke et al. J. Clin. Invest. 106:923 (2000);Ringquist et al., Cytometry 33:394 (1998)). This reversal can be used toexpedite clearance of the detectable moiety from a patient's system.

Modulators of the invention can also be used in in vitro settings toenhance or inhibit the effect of a nucleic acid ligand (e.g., aptamer)on a target molecule. For example, modulators of the invention can beused in target validation studies. Using modulators of the invention, itis possible to confirm that a response observed after inhibiting atarget molecule (with a nucleic acid ligand) is due to specificallyinhibiting that molecule.

The modulators of the invention can be formulated into pharmaceuticalcompositions that can include, in addition to the modulator, apharmaceutically acceptable carrier, diluent or excipient. The precisenature of the composition will depend, at least in part, on the natureof the modulator and the route of administration. Optimum dosingregimens can be readily established by one skilled in the art and canvary with the modulator, the patient and the effect sought. Generally,the modulator is administered IV, IM, IP, SC, orally or topically, asappropriate.

In another embodiment, the nucleic acid ligand or its regulator can becovalently attached to a lipophilic compound such as cholesterol,dialkyl glycerol, diacyl glycerol, or a non-immunogenic, high molecularweight compound or polymer such as polyethylene glycol (PEG). In thesecases, the pharmacokinetic properties of the nucleic acid ligand ormodulator can be enhanced. In still other embodiments, the nucleic acidligand or the modulator can be comprised for example, of a nucleic acidor PNA or MNA encapsulated inside a liposome, and the enhancedintracellular uptake is seen over the un-complexed oligonucleotide ormodulator. The lipophilic compound or non-immunogenic, high molecularweight compound can be covalently bonded or associated throughnon-covalent interactions with ligand or modulator(s). In embodimentswhere the lipophilic compound is cholesterol, dialkyl glycerol, diacylglycerol, or the non-immunogenic, high molecular weight compound is PEG,a covalent association with the oligonucleotide modulator (s) ispreferred. In embodiments where the lipophilic compound is a cationicliposome or where the oligonucleotide modulators are encapsulated withinthe liposome, a non-covalent association with the oligonucleotidemodulator (s) is preferred. In embodiments where covalent attachment isemployed, the lipophilic compound or non-immunogenic, high molecularweight compound may be covalently bound to a variety of positions on theoligonucleotide modulator, such as to an exocyclic amino group on thebase, the 5-position of a pyrimidine nucleotide, the 8-position of apurine nucleotide, the hydroxyl group of the phosphate, or a hydroxylgroup or other group at the 5′ or 3′ terminus of the oligonucleotidemodulator. Preferably, however, it is bonded to the 5′ or 3′ hydroxylgroup thereof. Attachment of the oligonucleotide modulator to othercomponents of the complex can be done directly or with the utilizationof linkers or spacers. The lipophilic compound or non-immunogenic, highmolecular weight compound can associate through non-covalentinteractions with the oligonucleotide modulator(s). For example, in oneembodiment of the present invention, the oligonucleotide modulator isencapsulated within the internal compartment of the lipophilic compound.In another embodiment of the present invention, the oligonucleotidemodulator associates with the lipophilic compound through electrostaticinteractions. For instance, a cationic liposome can associate with ananionic oligonucleotide modulator. Another example of a non-covalentinteraction through ionic attractive forces is one in which a portion ofthe oligonucleotide modulator hybridizes through Watson-Crickbase-pairing or triple helix base pairing to an oligonucleotide which isassociated with a lipophilic compound or non-immunogenic, high molecularweight compound.

I. Definitions

The following terms are believed to have well-recognized meanings in theart. However, the following definitions are set forth to facilitateexplanation of the invention.

The terms “binding activity” and “binding affinity’ are meant to referto the tendency of a ligand molecule to bind or not to bind to a target.The energy of said interactions are significant in “binding activity”and “binding affinity” because they define the necessary concentrationsof interacting partners, the rates at which these partners are capableof associating, and the relative concentrations of bound and freemolecules-in a solution. The energetics are characterized through, amongother ways, the determination of a dissociation constant, K_(d).Preferably, the K_(d) is established using a double-filternitrocellulose filter binding assay such as that disclosed by Wong andLohman, 1993, Proc. Natl. Acad. Sci. USA 90, 5428-5432. “Specificallybinding oligonucleotides”, “nucleic acid ligands” or “aptamers” in oneembodiment of the invention are oligonucleotides having specific bindingregions that are capable of forming complexes with an intended targetmolecule. The specificity of the binding is defined in terms of thecomparative dissociation constants (K_(d)) of a modulator of a nucleicacid ligand as compared to the dissociation constant with respect toother materials in the environment or unrelated molecules in general.The K_(d) for a modulator of a nucleic acid ligand can be 2-fold,preferably 5-fold, more preferably 10-fold less than the K_(d) withrespect to the modulator and the unrelated material or accompanyingmaterial in the environment. Even more preferably the K_(d) will be50-fold less, more preferably 100-fold less, and more preferably200-fold less.

K_(d) can be determined directly by well-known methods, and can becomputed even for complex mixtures by methods such as those, forexample, set forth in Caceci, M., et al., Byte {1984) 9:340-362. It hasbeen observed, however, that for some small oligonucleotides, directdetermination of K_(d) is difficult, and can lead to misleadingly, highresults. Under these circumstances, a competitive binding assay for thetarget molecule or other candidate substance can be conducted withrespect to substances known to bind the target or candidate. The valueof the concentration at which 50% inhibition occurs (K_(i)) is, underideal conditions, equivalent to K_(d). However, in no event will a K_(i)be less than K_(d). Thus, determination of K_(i), in the alternative,sets a maximal value for the value of K_(d). Under those circumstanceswhere technical difficulties preclude accurate measurement of K_(d),measurement of K_(i) can conveniently be substituted to provide an upperlimit for K_(d). A K_(i) value can also be used to confirm that amodulator binds a nucleic acid ligand.

As specificity is defined in terms of K_(d) as set forth above, incertain embodiments of the present invention it is preferred to excludefrom the categories of unrelated materials and materials accompanyingthe target in the target's environment those materials which aresufficiently related to the target to be immunologically cross-reactivetherewith. By “immunologically cross-reactive” is meant that antibodiesraised with respect to the target cross-react under standard assayconditions with the candidate material. Generally, for antibodies tocross-react in standard assays, the binding affinities of the antibodiesfor cross-reactive materials as compared to targets should be in therange of 5-fold to 100-fold, generally about 10-fold.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of compound need not be 100% complementary to that ofits target nucleic acid to be specifically hybridizable. A compound isspecifically hybridizable when binding of the compound to the targetnucleic acid molecule interferes with the normal function of the targetnucleic acid to cause a change in utility, and there is a sufficientdegree of complementarity to avoid non-specific binding of the compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed.

“Oligomers” or oligonucleotides” include RNA or DNA sequences ormixtures or analogs thereof, of more than one nucleotide in eithersingle chain or duplex form and specifically includes short sequencessuch as dimers and trimers, in either single chain or duplex form, whichcan be intermediates in the production of the specifically bindingoligonucleotides. “Modified” forms used in candidate pools contain atleast one non-native residue.

The term “RNA analog” is meant to refer to a polymeric molecule whichcan contain one or more nucleotides that have a nonhydrogen substituentother than a hydroxyl group in the 2′-position, and for example, cancontains at least one of the following: 2′-deoxy, 2′-halo (including2′-fluoro), 2′-amino (preferably not substituted or mono- ordisubstituted), 2′-mono-, di- or tri-halomethyl, 2′-O-alkyl (including2′-O-methyl or O-ethyl), 2′-O-halo-substituted alkyl, 2′-alkyl, azido,phosphorothioate, sulflhydryl, methylphosphonate, fluorescein,rhodamine, pyrene, biotin, xanthine, hypoxanthine, 2,6-diamino purine,2-hydroxy-6-mercaptopurine and pyrimidine bases substituted at the6-position with sulfur or 5 position with halo or C₁₋₅ alkyl groups, abasic linkers, 3′-deoxy-adenosine as well as other available “chainterminator” or “non-extendible” analogs (at the 3′-end of the RNA), orlabels such as ³²P, ³³P and the like. All of the foregoing can beincorporated into an RNA using the standard synthesis techniquesdisclosed herein.

As used herein, a “target” or “target molecule” refers to a biomoleculethat is the focus of a therapeutic drug strategy or diagnostic assay,including, without limitation, enzymes, enzyme inhibitors, hormones,glycoproteins, lipids, phospholipids, nucleic acids, intracellular,extracellular, and cell surface proteins, peptides, carbohydrates,including glycosaminoglycans, lipids, including glycolipids and certainoligonucleotides, and generally, any biomolecule capable of turning abiochemical pathway on or off or modulating it, or which is involved ina predictable biological response. Targets can be free in solution, likethrombin, or associated with cells or viruses, as in receptors orenvelope proteins. Any molecule that is of sufficient size to bespecifically recognized by a nucleic acid ligand can be used as thetarget. Thus, membrane structures, receptors, organelles, and the likecan be used as the complexation targets.

An “RNA aptamer” is an aptamer comprising ribonucleoside units. “RNAaptamer” is also meant to encompass RNA analogs as defined herein above.

The term “coagulation factor aptamer” is meant to refer to a single- ordouble-stranded nucleic acid that binds a coagulation factor andmodulates its function. The term “coagulation factor” is meant to referto a factor that acts in either or both of the intrinsic and theextrinsic coagulation cascade.

As used herein, “consensus sequence” refers to a nucleotide sequence orregion (which might or might not be made up of contiguous nucleotides)that is found in one or more regions of at least two nucleic acidsequences. A consensus sequence can be as short as three nucleotideslong. It also can be made up of one or more noncontiguous sequences,with nucleotide sequences or polymers of up to hundreds of bases longinterspersed between the consensus sequences. Consensus sequences can beidentified by sequence comparisons between individual nucleic acidspecies, which comparisons can be aided by computer programs and other,tools for modeling secondary and tertiary structure from sequenceinformation. Generally, the consensus sequence will contain at leastabout 3 to 20 nucleotides, more commonly from 6 to 10 nucleotides.

The terms “cardiovascular disease” and “cardiovascular diseases” aremeant to refer to any cardiovascular disease as would be understood byone of ordinary skill in the art. Nonlimiting examples of particularlycontemplated cardiovascular diseases include, but are not limited to,atherosclerosis, thrombophilia, embolisms, cardiac infarction (e.g.,myocardial infarction), thromboses, angina, stroke, septic shock,hypertension, hyper-cholesterolemia, restenosis and diabetes.

The term “about,” as used herein when referring to a measurable valuesuch as an amount of weight, time, dose etc. is meant, to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified amount, as suchvariations are appropriate to perform the disclosed method.

Compounds of the present invention having a chiral center may exist inand be isolated in optically active and racemic forms. Some compoundsmay exhibit polymorphism. The present invention encompasses racemic,optically-active, polymorphic, or stereoisomeric form, or mixturesthereof, of a compound of the invention, which possess the usefulproperties described herein. The optically active forms can be preparedby, for example, resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase or by enzymatic resolution. In a preferred embodiment,the compounds are used in naturally occurring forms. However,alternatively, the compounds can be used in a non-naturally occurringform.

II. Nucleic Acid Ligands

A “Nucleic Acid Ligand” (sometimes also referred to as an “aptamer”) asused herein is a non-naturally occurring nucleic acid having a desirableaction on a target. A desirable action includes, but is not limited to,binding of the target, catalytically changing the target, reacting withthe target in a way which modifies/alters the target or the functionalactivity of the target, covalently attaching to the target as in asuicide inhibitor, or facilitating the reaction between the target andanother molecule. In the preferred embodiment, the action is specificbinding affinity for a target molecule, such target molecule being athree dimensional chemical structure, wherein the nucleic acid ligand isnot a nucleic acid having the known physiological function of beingbound by the target molecule. In one embodiment of the invention, thenucleic acid ligands are identified using the SELEX methodology. Nucleicacid ligands includes nucleic acids that are identified from a candidatemixture of nucleic acids, wherein the nucleic acid ligand being a ligandof a given target by the method comprising a) contacting the candidatemixture with the target, wherein nucleic acids having an increasedaffinity to the target relative to the candidate mixture may bepartitioned from the remainder of the candidate mixture; b) partitioningthe increased affinity nucleic acids from the remainder of the candidatemixture; and c) amplifying the increased affinity nucleic acids to yielda ligand-enriched mixture of nucleic acids. As used herein nucleic acidligand or aptamer denotes both singular and plural sequences of nucleicacids which are capable of binding to a protein or other molecule, andthereby disturbing the protein's or other molecule's function.

Nucleic acid ligands can be made with nucleotides bearing D or Lstereochemistry, or a mixture thereof. Naturally occurring nucleosidesare in the D configuration. Aptamers have been made from L nucleotides,and are called L-aptamers. Nucleic acid ligands used for therapeuticpurposes are typically in the D configuration, but can exhibit anyconfiguration that provides the desired effect. Typically, when nucleicacid ligands comprising L-nucleotides are the target of anoligonucleotide modulator, the modulator also comprises L-nucleotides(see, for example, U.S. Pat. No. 5,780,221).

The nucleic acid ligands preferably comprise about 10 to about 100nucleotides, preferably about 15 to about 40 nucleotides, morepreferably about 20 to about 40 nucleotides, in that oligonucleotides ofa length that falls within these ranges are readily prepared byconventional, techniques. In one embodiment, aptamers or oligonucleotidemodulators can comprise a minimum of approximately 6 nucleotides,preferably 10, and more preferably 14 or 15 nucleotides, that arenecessary to effect specific binding. The only apparent limitations onthe binding specificity of the target/oligonucleotide couples of theinvention concern sufficient sequence to be distinctive in the bindingoligonucleotide and sufficient binding capacity of the target substanceto obtain the necessary interaction. Aptamers of binding regionscontaining sequences shorter than 10, e.g., 6-mers, are feasible if theappropriate interaction can be obtained in the context of theenvironment in which the target is placed. Thus, if there is littleinterference by other materials, less specificity and less strength ofbinding can be required.

Nucleic acid ligands and methods for their production and use, aredescribed, for example, in the following U.S. patents. Any of thenucleic acid ligands described in the patents listed below or otherpatents, or any nucleic acid ligands described in publications as wellas other desired nucleic acid ligands used in medical therapy can bemodulated or regulated according to the present invention. U.S. Pat. No.6,387,635, entitled 2′-fluoropyrimidine anti-calf intestinal phosphatasenucleic acid ligands; U.S. Pat. No. 6,387,620, entitledTranscription-free selex; U.S. Pat. No. 6,379,900, entitled Compositionsand methods of use of 8-nitroguanine; U.S. Pat. No. 6,376,474, entitledSystematic evolution of ligands by exponential enrichment: tissue SELEX;U.S. Pat. No. 6,376,190, entitled Modified SELEX processes withoutpurified protein; U.S. Pat. No. 6,355,787, entitled Purine nucleosidemodifications by palladium catalyzed methods and compounds produced;U.S. Pat. No. 6,355,431, entitled Detection of nucleic acidamplification reactions using bead arrays; U.S. Pat. No. 6,346,611,entitled High affinity TGFβ nucleic acid ligands and inhibitors; U.S.Pat. No. 6,344,321, entitled Nucleic acid ligands which bind tohepatocyte growth factor/scatter factor (HGF/SF) or its receptor c-met;U.S. Pat. No. 6,344,318, entitled Methods of producing nucleic acidligands; U.S. Pat. No. 6,331,398, entitled Nucleic acid ligands; U.S.Pat. No. 6,331,394, entitled Nucleic acid ligands to integrins; U.S.Pat. No. 6,329,145, entitled Determining non-nucleic acid moleculebinding to target by competition with nucleic acid ligand; U.S. Pat. No.6,306,598, entitled Nucleic acid-coupled colorimetric analyte detectors;U.S. Pat. No. 6,303,316, entitled Organic semiconductor recognitioncomplex and system; U.S. Pat. No. 6,300,074, entitled Systematicevolution of ligands by exponential enrichment: Chemi-SELEX; U.S. Pat.No. 6,291,184, entitled Systematic evolution of ligands by exponentialenrichment: photoselection of nucleic acid ligands and solution selex;U.S. Pat. No. 6,287,765, entitled Methods for detecting and identifyingsingle molecules; U.S. Pat. No. 6,280,943, entitled 2′-fluoropyrimidineanti-calf intestinal phosphatase nucleic acid ligands; U.S. Pat. No.6,280,932, entitled High affinity nucleic acid ligands to lectins; U.S.Pat. No. 6,264,825, entitled Binding acceleration techniques for thedetection of analytes; U.S. Pat. No. 6,261,783, entitled Homogeneousdetection of a target through nucleic acid ligand-ligand beaconinteraction; U.S. Pat. No. 6,261,774, entitled Truncation selex method;U.S. Pat. No. 6,242,246, entitled Nucleic acid ligand diagnosticBiochip; U.S. Pat. No. 6,232,071, entitled Tenascin-C nucleic acidligands; U.S. Pat. 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III. Types of Modulators

Modulators (i.e., regulators) of the invention include anypharmaceutically acceptable agent that can bind a nucleic acid ligandand modify the interaction between that ligand and its target molecule(e.g., by modifying the structure of the aptamer) in a desired manner,or which degrades, metabolizes, cleaves or otherwise chemically altersthe nucleic acid ligand to modify its biological effect. Examples ofmodulators include (A) oligonucleotides or analogues thereof that arecomplementary to at least a portion of the aptamer sequence (includingribozymes or DNAzymes or, for example, peptide nucleic acids (PNAs),mopholino nucleic acids (MNAs), or Locked Nucleic Acids (LNAs)), (B)nucleic acid binding peptides, polypeptides or proteins (includingnucleic acid binding tripeptides (see, generally, Hwang et al., Proc.Natl. Acad. Sci. USA 96:12997 (1999)), (C) oligosaccharides (e.g.aminoglycosides (see, generally, Davis et al., Chapter 8, p. 185, RNAWorld, Cold Spring Harbor Laboratory Press, eds Gestlaad and Atkins(1993), (D) small molecules, and (E) chimeras, fusion products, or othercombinations of any of the above. Werstuck et al., Sciende 282:296(1998), USPs 5,935,776 and 5,534,408). (See also the following whichdisclose types of modulators that can be used in accordance with thepresent invention: Chase et al., Ann. Rev. Biochem. 56:103 (1986),Eichorn et al., J. Am. Chem. Soc. 90:7323 (1968), Dale et al.,Biochemistry 14:2447 (1975) and Lippard et al., Acc. Chem. Res. 11:211(1978)).

In an alternative embodiment of the invention, the modulator itself isan aptamer. In this embodiment, a nucleic acid ligand is first generatedthat binds to the desired therapeutic target. In a second step, a secondaptamer that binds to the first aptamer is generated using the SELEXprocess described herein or other process, and modulates the interactionbetween the therapeutic aptamer and the target.

In other alternative embodiments, the nucleic acid ligand which binds tothe target is a PNA, MNA, LNA or PCO and the modulator is an aptamer.Alternatively, the nucleic acid ligand which binds to the target is aPNA, MNA, LNA or PCO, and the modulator is an MNA. Alternatively, thenucleic acid ligand which binds to the target is a PNA, MNA, LNA or PCO,and the modulator is an LNA. Alternatively, the nucleic acid ligandwhich binds to the target is a PNA, MNA, LNA or PCO, and the modulatoris a PCO.

Oligonucleotides and Analogues Thereof

1. Polynucleic Acid

In a preferred embodiment, the modulator of the invention is anoligonucleotide that comprises a sequence complementary to at least aportion of the targeted nucleic acid ligand sequence. For example, themodulator oligonucleotide can comprise a sequence complementary to 6-25nucleotides of the targeted nucleic acid ligand, typically, 8-20nucleotides, more typically, 10-15 nucleotides. Advantageously, themodulator oligonucleotide is complementary to 6-25 consecutivenucleotides of the nucleic acid ligand, or 8-20 or 10-15 consecutivenucleotides. The length of the modulator oligonucleotide can be readilyoptimized taking into account the targeted nucleic acid ligand and theeffect sought. Typically the modulator oligonucleotide is 5-80nucleotides in length, more typically, 10-30 and most typically 15-20nucleotides (e.g., 15-17). The oligonucleotide can be made withnucleotides bearing D or L stereochemistry, or a mixture thereof.Naturally occurring nucleosides are in the D configuration.

Formation of duplexes by binding of complementary pairs of shortoligonucleotides is a fairly rapid reaction with second orderassociation rate constants generally between 1×10⁶ and 3×10⁵ M¹s¹. Theinitial phase of the binding reaction involves formation of a two tothree base pair duplex, termed “nucleation”. After nucleation, pairingproceeds through the entire length of the complementary sequence at avery fast rate—approximately 10⁶ bases per second at 20° C. Thus, themodulatory effect on a nucleic acid ligand by formation of a duplex witha complimentary oligonucleotide is rapid. Stability of short duplexes ishighly dependent on the length and base-composition of the duplex. Thethermodynamic parameters for formation of short nucleic acid duplexeshave been rigorously measured, resulting in nearest-neighbor rules forall possible base pairs such that accurate predictions of the freeenergy, T_(m) and thus half-life of a given oligoribonucleotide duplexcan be calculated (e.g., Xia et al., Biochem. 37:14719 (1998) (see alsoEguchi et al. Antigensis RNA, Annu. Rev. Biochem. 60:631 (1991)).

Oligonucleotide modulators of the invention are advantageously targetedat single-stranded regions of the nucleic acid ligand. This facilitatesnucleation and, therefore, the rate of nucleic acid ligand activitymodulation, and also, generally leads to intermolecular duplexes thatcontain more base pairs than the targeted nucleic acid ligand.

Various strategies can be used to determine the optimal site foroligonucleotide binding to a targeted nucleic acid ligand. An empiricalstrategy can be used in which complimentary oligonucleotides are“walked” around the aptamer. A walking experiment can involve twoexperiments performed sequentially. A new candidate mixture can beproduced in which each of the members of the candidate mixture has afixed nucleic acid-region that corresponds to a oligonucleotidemodulator of interest. Each member of the candidate mixture alsocontains a randomized region of sequences. According to this method itis possible to identify what are referred to as “extended” nucleic acidligands, which contain regions that can bind to more than one bindingdomain of an nucleic acid ligand. In accordance with this approach,2′-O-methyl oligonucleotides (e.g., 2′-O-methyl oligonucleotides) about15 nucleotides in length can be used that are staggered by about 5nucleotides on the aptamer (e.g., oligonucleotides complementary tonucleotides 1-15, 6-20, 11-25, etc. of aptamer 9.3t. An empiricalstrategy can be particularly effective because the impact of thetertiary structure of the aptamer on the efficiency of hybridization canbe difficult to predict. Assays described in the Examples that followcan be used to assess the ability of the different oligonucleotides tohybridize to a specific nucleic acid ligand, with particular emphasis onthe molar excess of the oligonucleotide required to achieve completebinding of the nucleic acid ligand. The ability of the differentoligonucleotide modulators to increase the rate of dissociation of thenucleic acid ligand from, or association of the nucleic acid ligandwith, its target molecule can also be determined by conducting standardkinetic studies using, for example, BIACORE assays. Oligonucleotidemodulators can be selected such that a 5-50 fold molar excess ofoligonucleotide, or less, is required to modify the interaction betweenthe nucleic acid ligand and its target molecule in the desired manner.

Alternatively, the targeted nucleic acid ligand can be modified so as toinclude a single-stranded tail (3′ or 5′) in order to promoteassociation with an oligonucleotide modulator. Suitable tails cancomprise 1 to 20 nucleotides, preferably, 1-10 nucleotides, morepreferably, 1-5 nucleotides and, most preferably, 3-5 nucleotides (e.g.,modified nucleotides such as 2′-O-methyl sequences). Tailed nucleic acidligands can be tested in binding and bioassays (e.g., as described inthe Examples that follow) to verify that addition of the single-strandedtail does not disrupt the active structure of the nucleic acid ligand. Aseries of oligonucleotides (for example, 2′-O-methyl oligonucleotides)that can form, for example, 1, 3 or 5 base pairs with the tail sequencecan be designed and tested for their ability to associate with thetailed aptamer alone, as well as their ability to increase the rate ofdissociation of the nucleic acid ligand from, or association of theaptamer with, its target molecule. Scrambled sequence controls can beemployed to verify that the effects are due to duplex formation and notnon-specific effects. The native gel assay described in the Examplesthat follow can be used to measure the dissociation/association rate ofan oligonucleotide modulator from/with a target aptamer.

As an illustrative embodiment, the present invention provides modulatorsthat specifically and rapidly reverse the anticoagulant andantithrombotic effects of nucleic acid ligands that target components ofthe coagulation pathway, particularly nucleic acid ligand antagonists ofthe tissue factor (TF)/factor VIIa (FVIIa), factor VIIIa (FVIIIa)/factorIXa (FIXa), factor Va (FVa/factor Xa (Fxa) enzyme complexes and plateletreceptors such as gpIIbIIIa, gpIbIX, gpVI, factors involved in promotingplatelet activation such as Gas6, factors involved in promoting ormaintaining fibrin clot formation such as PAI-1 (plasminogen activatorinhibitor 1) or coagulation factor XIIIa (FXIIIa), and additionalfactors involved in promoting or preventing fibrin clot formation suchas ATIII (anti-thrombin III), thrombin or coagulation factor XIa (FXIa).In accordance with this embodiment, modulators (in this case,inhibitors, advantageously, oligonucleotide inhibitors) are administeredthat reverse the nucleic acid ligand activity.

In specific embodiments, modulators of nucleic acid ligand activityaccording to the present invention are nucleic acids selected from thegroup consisting of, but not limited to the following sequences;5′AUGGGGAGGCAGCAUUA 3′ (SEQ ID NO:25), 5′CAUGGGGAGGCAGCAUUA3′ (SEQ IDNO:26), 5′CAUGGGGAGGCAGCA3′ (SEQ ID NO:27), 5′CAUGGGGAGGCA3′ (SEQ IDNO:28), 5′GCAUUACGCGGUAUAGUCCCCUA3′ (SEQ ID NO:29),5′CGCGGUAUAGUCCCCUA3′ (SEQ ID NO:30), 5′CUCGCUGGGGCUCUC3′ (SEQ ID NO:31), 5′UAUUAUCUCGCUGGG3′ (SEQ ID NO:32), 5′AAGAGCGGGGCCAAG3′ (SEQ IDNO:33), 5′GGGCCAAGUAUUAU 3′ (SEQ ID NO:34), 5′CAAGAGCGGGGCCAAG 3′ (SEQID NO:35), 5′CGAGUAUUAUCUUG3′ (SEQ ID NO:36), 5′CGCGGUAUAGUCCCCAU3′ (SEQID NO:41), or any modification or derivative thereof in whichhybridization is maintained or is optionally at least 95% homologous tothe sequence.

For example, the inhibitor of a nucleic acid ligand to Factor IXa of thepresent invention can be an antisense oligonucleotide. The antisenseoligonucleotide hybridizes to the nucleic acid ligand in vivo and blocksthe binding of the nucleic acid ligand to factor IXa.

The oligonucleotide modulators of the invention comprise a sequencecomplementary to at least a portion of a nucleic acid ligand. However,absolute complementarity is not required. A sequence “complementary toat least a portion of a nucleic acid ligand,” referred to herein, meansa sequence having sufficient complementarity to be able to hybridizewith the nucleic acid ligand. The ability to hybridize can depend onboth the degree of complementarity and the length of the antisensenucleic acid. Generally, the larger the hybridizing oligonucleotide, themore base mismatches with a target nucleic acid ligand it can containand still form a stable duplex (or triplex as the case may, be). Oneskilled in the art can ascertain a tolerable degree of mismatch by useof standard procedures to determine the melting point of the hybridizedcomplex. In specific aspects, the oligonucleotide can be at least 5 orat least 10 nucleotides, at least 15 or 17 nucleotides, at least 25nucleotides or at least 50 nucleotides. The oligonucleotides of theinvention can be DNA or RNA or chimeric mixtures or derivatives ormodified versions thereof, single-stranded.

Antisense techniques are discussed for example, in Okano, J.,Neurochein. 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988). The methods arebased on binding of a polynucleotide to a complementary DNA or RNA.

Oligonucleotides of the invention can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide can includeother appended groups. To this end, the oligonucleotide can beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, hybridization-triggered cleavageagent, etc. The oligonucleotide can comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine,N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine,N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2α-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N&isopentenyladenine, uracil oxyacetic acid (y),wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methylthiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,-uracil-5-oxyacetic acid methylester, uracil oxyacetic acid (v),5-methyl thiouracil, 3-(3-amino-3-N carboxypropyl) and2,6-diaminopurine.

An oligonucleotide modulator of the invention can also comprise at leastone modified sugar moiety selected from the group including, but notlimited to, arabinose, 2-fluoroarabinose, xylose, and hexose.

In yet another embodiment, an oligonucleotide modulator can comprise atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphorodiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

Oligonucleotide modulators can be prepared that have desiredcharacteristics, such as improved in vivo stability and/or improveddelivery characteristics. Examples of such modifications includechemical substitutions at the sugar and/or backbone and/or basepositions. Oligonucleotide modulators can contain nucleotide derivativesmodified at the 5- and 2′ positions of pyrimidines, for example,nucleotides can be modified with 2′ amino, 2′-fluoro and/or 2′-O-methyl.Modifications of the modulating oligonucleotides of the invention caninclude those that provide other chemical groups that incorporateadditional charge, polarization, hydrophobicity, hydrogen bonding and/orelectrostatic interaction. Such modifications include but are notlimited to 2′ position sugar modifications, locked nucleic acids, 5position pyrimidine modifications, 8 position purine modifications,modification at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil, backbone modifications,phosphorothioate or alkyl phosphate modifications, methylations, unusualbase-pairing combinations such as isobases isocytidine and isoguanidine,etc. Modifications can also include 3′ and 5′ modifications, such ascapping. (See also Manoharan, Biochem. Biophys. Acta 1489:117 (1999);Herdewija, Antisense Nucleic Acid Drug Development 10:297 (2000); Maieret al., Organic Letters 2:1819 (2000)).

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.).

Using the instructions provided herein, one of ordinary skill can easilypractice the invention to regulate any therapeutic nucleic acid by thetimely administration of a complementary nucleic acid that terminates orotherwise modifies the activity of the therapeutic ligand. Thistechnique can be used, for example, in connection with any of thenucleic acid ligands described or referred to in the patent documentscited above.

2. Ribozymes and DNAzymes

Likewise, using the instructions provided herein, one of ordinary skillcan easily practice the invention to regulate any therapeutic nucleicacid ligand by the timely administration of a ribozyme or a DNAzyme.

Enzymatic nucleic acids act by first binding to a target RNA (or DNA,see Cech U.S. Pat. No. 5,180,818). Such binding occurs through thetarget binding portion of an enzymatic nucleic acid which is held inclose proximity to an enzymatic portion of molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Cleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targetsthereby allowing for inactivation of RNA aptamers. There are at leastfive classes of ribozymes that each display a different type ofspecificity. For example, Group I Introns are ˜300 to >1000 nucleotidesin size and require a U in the target sequence immediately 5′ of thecleavage site and binds 4-6 nucleotides at the 5′-side of the cleavagesite. There are over 75 known members of this class, they are found inTetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phageT4, blue-green algae, and others. Another class are RNaseP RNA (M1 RNA),which are ˜290 to 400 nucleotides in size. They are the RNA portion of aribonucleoprotein enzyme and cleave the tRNA precursors to form maturetRNA There are roughly 10 known members of this group and all arebacterial in origin. A third example are Hammerhead Ribozyme, which are˜30 to 40 nucleotides in size. They require the target sequence UHimmediately 5′ of the cleavage site and bind a variable numbernucleotides on both sides of the cleavage site. There are at least 14known members of this class that are found in a number of plantpathogens (virusoids) that use RNA as the infectious agent. A fourthclass are the Hairpin Ribozymes, which are ˜50 nucleotides in size. Theyrequires the target sequence GUC immediately 3′ of the cleavage site andbind 4 nucleotides at the 5′-side of the cleavage site and a variablenumber to the 3′-side of the cleavage site. There are found in one plantpathogen (satellite RNA of the tobacco ringspot virus) which uses RNA asthe infectious agent. The fifth group are Hepatitis Delta Virus (HDV)Ribozymes, which are ˜60 nucleotides in size.

The enzymatic nature of a ribozyme can be advantageous over othertechnologies, such as antisense technology, in certain applicationssince the effective concentration of ribozyme necessary to effect atherapeutic treatment can be less than that of an antisenseoligonucleotide. This advantage reflects the ability of the ribozyme toact enzymatically. Thus, a single ribozyme molecule is able to cleavemany molecules of target RNA aptamers. In addition, the ribozyme is ahighly specific inhibitor, with the specificity of inhibition dependingnot only on the base pairing mechanism of binding, but also on themechanism by which the molecule inhibits the expression of the RNA towhich it binds. That is, the inhibition is caused by cleavage of the RNAtarget and so specificity is defined as the ratio of the rate ofcleavage of the targeted RNA over the rate of cleavage of non-targetedRNA. This cleavage mechanism is dependent upon factors additional tothose involved in base pairing. Thus, the specificity of action of aribozyme, under certain circumstances, can be greater than that ofantisense oligonucleotide binding the same RNA site.

Another class of catalytic molecules are called “DNAzymes”. DNAzymes aresingle-stranded, and cleave both RNA and DNA. A general model for theDNAzyme has been proposed, and is known as the “10-23” model. DNAzymesfollowing the “10-23” model, also referred to simply as “10-23DNAzymes”, have a catalytic domain of 15 deoxyribonucleotides, flankedby two substrate-recognition domains of seven to ninedeoxyribonucleotides each. In vitro analyses show that this type ofDNAzyme can effectively cleave its substrate RNA at purine:pyrimidinejunctions under physiological conditions. As used herein, “DNAzyme”means a DNA molecule that specifically recognizes and cleaves a distincttarget nucleic acid sequence, which may be either DNA or RNA.

Ribozymes and DNAzymes comprise both substrate binding domains andcatalytic domains. Rules for the optimal design of the regulatoryribozymes can be found within Methods in Molecular Biology Volume 74“Ribozyme Protocols” edited by Philip C. Turner (Humana Press, Totowa,N.J., 1997). Strategies to identify reactive sites for ribozymes withina targeted nucleic acid are also described in this volume. Using thesestandard rules, hammerhead ribozymes, hairpin ribozymes, hepatitis deltavirus ribozymes and ribozymes derived from group I introns can bespecifically engineered to bind to and cleave a target nucleic acidligand. The ability of such ribozymes or DNAzymes to cleave the targetnucleic acid ligand can be determined in any number of assays. Forexample, following incubation of a ribozyme or DNAzyme with an unlabeledor a labeled (eg. ³²P) target nucleic acid ligand under conditionsfavoring reaction, the target nucleic acid can be analyzed by denaturinggel electrophoresis to determine if the ribozyme or DNAzyme cleaved thebackbone of the target nucleic acid ligand Alternatively, fluorescenceresonance energy transfer, or change in fluorescence intensity can beused to measure cleavage of an appropriately labeled nucleic acidligand.

3. Polynucleic Acid Analogues (Peptide Nucleic Acids (PNAs), MorpholinoNucleic Acids (MNAs), Locked Nucleic Acids (LNAs)), and Pseudo-CyclicOligonucleotides (PCOs)

Nucleobases of the oligonucleotide modulators of the invention can beconnected via internucleobase linkages, e.g., peptidyl linkages (as inthe case of peptide nucleic acids (PNAs); Nielsen et al. (1991) Science254, 1497 and U.S. Pat. No. 5,539,082) and morpholino linkages (Qin etal., Antisense Nucleic Acid Drug Dev. 10, 11 (2000); Summerton,Antisense Nucleic Acid Drug Dev. 7, 187 (1997); Summerton et al.,Antisense Nucleic Acid Drug Dev. 7, 63 (1997); Taylor et al., J Biol.Chem. 271, 17445 (1996); Partridge et al., Antisense Nucleic Acid DrugDev. 6, 169 (1996)), or by any other natural or modified linkage. Theoligonucleobases can also be Locked Nucleic Acids (LNAs). Nielsen etal., J Biomol Struct Dyn 17, 175 (1999); Petersen et al., J Mol Recognit13, 44 (2000); Nielsen et al., Bioconjug Chem 11, 228 (2000).

PNAs are compounds that are analogous to oligonucleotides, but differ incomposition. In PNAs, the deoxyribose backbone of oligonucleotide isreplaced with a peptide backbone. Each subunit of the peptide backboneis attached to a naturally-occurring or non-naturally-occurringnucleobase. PNA often has an achiral polyamide backbone consisting ofN-(2-aminoethyl)glycine units. The purine or pyrimidine bases are linkedto each unit via a methylene carbonyl linker (1-3) to target thecomplementary nucleic acid. PNA binds to complementary RNA or DNA in aparallel or antiparallel orientation following the Watson-Crickbase-pairing rules. The uncharged nature of the PNA oligomers enhancesthe stability of the hybrid PNA/DNA(RNA) duplexes as compared to thenatural homoduplexes. The non-natural character of the PNA makes PNAoligomers highly resistant to protease and nuclease attacks. Theseproperties of PNA oligomers allow them to serve as efficient antisenseor modulators of nucleic acid ligand activity. Indeed, peptide nucleicacids have been applied to block protein expression on thetranscriptional and translational level, and microinjected PNA oligomersdemonstrate a strong antisense effect in intact cells. Seewww.bioscience.org/1999/v4/d/soomets/fulltext.htm; and Frontiers inBioscience, 4, d782-786 (Nov. 1, 1999) for details on recentachievements on PNA antisense application, especially these concernedwith whole cell or tissue delivery of the PNA. See also Nielsen, P. E.,Egholm. M., Berg, R. H. & Buchardt, O. (1993) Peptide nucleic acids(PNA). DNA analogues with a polyamide backbone. In “Antisense Researchand Application” Crook, S. & Lebleu, B. (eds.) CRC Press, Boca Raton, pp363-373.

PNAs bind to both DNA and RNA and form PNA/DNA or PNA/RNA duplexes. Theresulting PNA/DNA or PNA/RNA duplexes are bound tighter thancorresponding DNA/DNA or DNA/RNA duplexes as evidenced by their highermelting temperatures (T_(m)). This high thermal stability ofPNA/DNA(RNA) duplexes has been attributed to the neutrality of the PNAbackbone, which results elimination of charge repulsion that is presentin DNA/DNA or RNA/RNA duplexes. Another advantage of PNA/DNA(RNA)duplexes is that T_(m) is practically independent of salt concentration.DNA/DNA duplexes, on the other hand, are highly dependent on the ionicstrength.

Since PNAs are an analogue of DNA in which the backbone is apseudopeptide rather than a sugar, they mimic the behavior of DNA andbind complementary nucleic acid strands. Unnatural nucleobases, such aspseudo isocytosine, 5-methyl cytosine and 2,6-diaminopurine, among manyothers, also can be incorporated in PNA synthons. PNAs are most commonlysynthesized from monomers (PNA synthons) protected according to thet-Boc/benzyl protection strategy, wherein the backbone amino group ofthe growing polymer is protected with the t-butyloxycarbonyl (t-Boc)group and the exocyclic amino groups of the nucleobases, if present, areprotected with the benzyloxycarbonyl (benzyl) group. PNA synthonsprotected using the t-Boc/benzyl strategy are now commerciallyavailable.

PNA is both biologically and chemically stable and readily available byautomated synthesis ((Hyrup, B., Egholm, M., Rolland, M., Nielsen, P.E., Berg, R. H. & Buchardt, O. (1993) Modification of the bindingaffinity of peptide nucleic acids (PNA). PNA with extended backbonesconsisting of 2-Aminoethyl-Alanine or 3-Aminopropylglycine units. J.Chem. Soc. Chem. Commun. 518-519); Demidov, V., Frank-Kamenetskii, M.D., Egholm, M., Buchardt, O. & Nielsen, P. E. (1993). Sequence selectivedouble strand DNA cleavage by PNA targeting using nuclease S1. NucleicAcids Res. 21, 2103-2107). These properties have made PNA very good leadfor antisense gene therapeutic drugs, and in vitro studies have furthersubstantiated the antisense potential (Nielsen, P. E. “Peptide NucleicAcid (PNA) A model structure for the primordial genetic material”Origins of Life 1993, 23, 323-327; Egholm, M., Behrens, C., Christensen,L., Berg, R. H., Nielsen, P. E. & Buchardt, O. “Peptide nucleic acidscontaining adenine or guanine recognize thymine and cytosine incomplementary DNA sequences” J. Chem. Soc. Chem. Commun. 1993, 800-801;Kim, S. K., Nielsen, P. E., Egholm, M., Buchardt, O., Berg, R. H. &Nordén, B. “Right-handed triplex formed between peptide nucleic acidPNA-T₈ and poly(dA) shown by linear and circular dichroism spectroscopy”J. Amer. Chem. Soc. 1993, 115, 6477-6481; Egholm, M., Buchardt, O.,Christensen, L., Behrens, C., Freier, S. M., Driver, D. A., Berg, R. H.,Kim, S. K., Nordén, B. & Nielsen, P. E. “PNA hybridizes to complementaryoligonucleotides obeying the Watson-Crick hydrogen bonding rules” Nature1993, 365, 556-568; Buchardt, O., Egholm, M., Berg, R. & Nielsen, P. E.“Peptide Nucleic Acids (PNA) and their potential applications inmedicine and biotechnology” Trends Biotechnology, 1993, 11, 384-386).

PNAs appear to be the very useful for a number of special applications.When targeted against rare all-purine sequences, PNAs can blocktranslation anywhere in a mRNA by forming a double-clamp structure. Withsuch rare RNA sequences one segment of the PNA binds to the targetsequence by Watson/Crick bonds and the other segment of the PNA binds tomajor-groove sites of the resulting PNA/RNA duplex. Probably because oftheir very flexible backbone structure, PNAs also readily form triplehelix structures with rare duplex DNA sequences comprising mostlypurines in one strand and mostly pyrimidines in the other strand.Lastly, under low salt conditions in cell-free systems PNAs have beenshown to achieve sequence-specific invasion of duplex DNA sequences,resulting in inhibition of transcription of the invaded duplex.

Recent studies by several groups have shown that coupling of PNA todifferent carriers can improve their uptake into cells. Among these,“cellular uptake peptides,” fatty acids or DNA, especially severalpeptide sequences have been shown to be able to carry PNA oligomersacross the cell membranes. Vector peptide-PNA conjugates have been shownto cross the neuron membrane and suppress targeted mRNA(Aldrian-Herrada, G. et al. (1998) Nucleic Acid Res. 26:4920).Biotinylated PNA linked to a conjugate of steptavidin and the OX26murine monoclonal antibody to the rat transferrin receptor have beenreported to cross the rat blood-brain barrier in vivo (Pardridge, W. etal. 1995 PNAS 92:5592-5596). Chinnery, P. F. et al. attached thepresequence peptide of the nuclear-encoded human cytochrome c oxidase(COX) subunit VIII to biotinylated PNA which was successfully importedinto isolated mitochondria in vitro (Chinnery, P. F. et al. 1999 GeneTher. 6:1919-28). Delivery of the biotinylated peptide-PNA tomitochondria in intact cells was confirmed by confocal microscopy. Inaddition, a short hydrophobic peptide with the sequence biotinyl-FLFLcoupled to a PNA trimer has been shown to internalize into humanerythrocytes and Namalwa cells (Scarfi, S., A. Gasparini, G. Damonte &U. Benatti: Synthesis, uptake, and intracellular metabolism of ahydrophobic tetrapeptide-peptide nucleic acid (PNA)-biotin molecule.Biochem Biophys Res Comnizin 1997, 236, 323-326). Basu and Wickström(Synthesis and characterization of a peptide nucleic acid conjugated toa D-peptide analog of insulin-like growth factor 1 for increasedcellular uptake. Bioconjugate Chem 1997, 8, 481-488) showed that PNAconjugated to an all-D-amino acid insulin-like growth factor 1 (IGF1)mimicking peptide was specifically taken up by cells expressing the IGF1receptor, although no antisense activity was described.

For some examples of other works, see Nielsen, P. E., M. Egholm, R. H.Berg & O. Buchardt: Sequence-selective recognition of DNA by stranddisplacement with a thymine-substituted polyamide. Science 254,1497-1500 (1991); Egholm, M., O. Buchart, P. E. Nielsen & R. H. Berg:Peptide nucleic acids (PNA). Oligonucleotide analogues with an achiralpeptide backbone. J Am Chem Soc 114, 1895-1897 (1992); Nielsen, P. E.,M. Egholm & O. Buchardt: Peptide nucleic acid (PNA). A DNA mimic with apeptide backbone. Bioconjugate Chemistry 5, 3-7 (1994); Egholm, M., P.E. Nielsen, O. Buchardt & R. H. Berg: Recognition of guanine and adeninein DNA by cytosine and thymine containing peptide nucleic acid. J AmChem Soc 114, 9677-9678 (1992); Egholm, M., O. Buchardt, L. Christensen,C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden& P. E. Nielsen: PNA hybridizes to complementary oligonucleotidesobeying the Watson-Crick hydrogen-bonding rules [see comments]. Nature365, 566-568 (1993); Wittung, P., P. E. Nielsen, O. Buchardt, M. Egholm& B. Nordén: DNA-like double helix formed by peptide nucleic acid.Nature 368, 561-563 (1994); Brown, S. C., S. A. Thomson, J. M. Veal & D.G. Davis: NMR solution structure of a peptide nucleic acid complexedwith RNA. Science 265, 777-780 (1994); Demidov, V. V., V. N. Potaman, M.D. Frank-Kamenetskii, M. Egholm, O. Buchard, S. H. Sönnichsen & P. E.Nielsen: Stability of peptide nucleic acids in human serum and cellularextracts. Biochemical Pharmacology 48, 1310-1313 (1994); Peffer, N. J.,J. C. Hanvey, J. E. Bisi, S. A. Thomson, C. F. Hassman, S. A. Noble & L.E. Babiss: Strand-invasion of duplex DNA by peptide nucleic acidoligomers. Proc Nat Acad Sci USA 90, 10648-10652 (1993).

U.S. Pat. No. 6,046,307 to Shay et al. discloses PNAs that inhibittelomerase activity in mammalian cells. U.S. Pat. No. 5,789,573 to Bakeret al. disclose compositions and methods for inhibiting the translationof capped targeted mRNA using PNAs. U.S. Pat. No. 6,165,720 discloselabeling of the PNA complex.

One can easily prepare a PNA or MNA that is complementary at least inpart to a nucleic acid ligand using the same rules of complementarityand Watson Crick base-pairing as is used in the preparation ofconventional oligonucleotide antisense molecules.

Morpholino nucleic acids are so named because they are assembled frommorpholino subunits, each of which contains one of the four geneticbases (adenine, cytosine, guanine, and thymine) linked to a 6-memberedmorpholine ring. Eighteen to twenty-five subunits of these four subunittypes are joined in a specific order by non-ionic phosphorodiamidateintersubunit linkages to give a morpholino oligo. These morpholinooligos, with their 6-membered morpholine backbone moieties joined bynon-ionic linkages, afford substantially better antisense propertiesthan do RNA, DNA, and their analogs having 5-membered ribose ordeoxyribose backbone moieties joined by ionic linkages (seewwwgene-tools.com/Morpholinos/body_morpholinos.HTML).

Morpholinos, devised by Summerton in 1985, constitute a radical redesignof natural nucleic acids, with the potential advantages of low coststarting materials and inexpensive assembly. Like PNAs, morpholinos arecompletely resistant to nucleases and they appear to be free of most orall of the non-antisense effects that are seen with S-DNAs. In contrastto PNAs, most morpholinos exhibit excellent aqueous solubility.Morpholinos also have much higher RNA binding affinities than do S-DNAs,though not as high as PNAs. Probably as a result of their substantialRNA binding affinities, long morpholinos (25-mers) provide predictabletargeting and very high efficacy. Most notable, morpholinos provide goodsequence specificity. The same factors that underlie their exceptionalsequence specificity also render them unsuitable for targeting pointmutations.

U.S. Pat. No. 6,153,737 to Manoharan et al. is directed to derivatizedoligonucleotides wherein the linked nucleosides are functionalized withpeptides, proteins, water soluble vitamins or lipid soluble vitamins.This disclosure was directed towards antisense therapeutics bymodification of oligonucleotides with a peptide or protein sequence thataids in the selective entry of the complex into the nuclear envelope.Similarly, water-soluble and lipid-soluble vitamins can be used toassist in the transfer of the anti-sense therapeutic or diagnostic agentacross cellular membranes.

The efficient and sequence specific binding to RNA or DNA combined withvery high biological stability makes PNAs and MNAs extremely attractiveleads for the development of modulators of the present invention.Peptide nucleic acids which can be conjugated to the carriers aredistinguished in U.S. Pat. No. 5,864,010 entitled Peptide Nucleic AcidCombinatorial Libraries and Improved Methods of Synthesis, developed byISIS Pharmaceuticals, which is hereby incorporated by reference. Inaddition, the peptide nucleic acids which can be conjugated to thecarriers of the present invention are distinguished in U.S. Pat. No.5,986,053 entitled peptide nucleic acids complexes of two peptidenucleic acid strands and one nucleic acid strand.

LNA is a novel class of DNA analogues that possess some features thatmake it a prime candidate for modulators of the invention. The LNAmonomers are bi-cyclic compounds structurally similar to RNA-monomers.LNA share most of the chemical properties of DNA and RNA, it iswater-soluble, can be separated by gel electrophoreses, ethanolprecipitated etc (Tetrahedron, 54, 3607-3630 (1998)). However,introduction of LNA monomers into either DNA or RNA oligos results inhigh thermal stability of duplexes with complementary DNA or RNA, while,at the same time obeying the Watson-Crick base-pairing rules. This highthermal stability of the duplexes formed with LNA oligomers togetherwith the finding that primers containing 3′ located LNA(s) aresubstrates for enzymatic extensions, e.g. the PCR reaction, is used inthe present invention to significantly increase the specificity ofdetection of variant nucleic acids in the in vitro assays described inthe application. The amplification processes of individual alleles occurhighly discriminative (cross reactions are not visible) and severalreactions may take place in the same vessel. See for example U.S. Pat.No. 6,316,198.

Pseudo-cyclic oligonucleobases (PCOs) can also be used as a modulator inthe present invention (see U.S. Pat. No. 6,383,752). PCOs contain twooligonucleotide segments attached through their 3′-3′ or 5′-5′ ends. Oneof the segments (the “functional segment”) of the PCO has somefunctionality (e.g., an antisense oligonucleotide complementary to atarget mRNA). Another segment (the “protective segment”) iscomplementary to the 3′- or 5′-terminal end of the functional segment(depending on the end through which it is attached to the functionalsegment). As a result of complementarity between the functional andprotective segment segments, PCOs form intramolecular pseudo-cyclicstructures in the absence of the target nucleic acids (e.g., RNA). PCOsare more stable than conventional antisense oligonucleotides because ofthe presence of 3′-3′ or 5′-5′ linkages and the formation ofintramolecular pseudo-cyclic structures. Pharmacokinetic, tissuedistribution, and stability studies in mice suggest that PCOs havehigher in vivo stability than and, pharmacokinetic and tissuedistribution profiles similar to, those of PS-oligonucleotides ingeneral, but rapid elimination from selected tissues. When a fluorophoreand quencher molecules are appropriately linked to the PCOs of thepresent invention, the molecule will fluoresce when it is in the linearconfiguration, but the fluorescence is quenched in the cyclicconformation.

A. Nucleic Acid Binding Peptides, Polypeptides or Proteins

Protein-nucleic acid interactions are involved in many cellularfunctions, including transcription, RNA splicing, and translation.Synthetic molecules that can bind with high affinity to specificsequences of single-stranded nucleic acids have the potential tointerfere with these interactions in a controllable way.

Many proteins have been identified that can bind directly to nucleicacid sequences. One main group of proteins that directly bind to DNA aretranscription factors. Some examples of transcription factors fall intothe following classes: Basic domain transcription factors(leucine-zipper factors, CREB, helix-loop-helix factors,helix-loop-helix/leucine zipper factors, cell-cycle controlling factors,NF-1, RF-X, bHSH), Zinc-coordinating DNA binding domain transcriptionfactors (cys4 zinc finger of nuclear receptor type, thyroid hormonereceptor-like factors, diverse Cys4 zinc fingers, Cys2His2 zinc fingerdomain, metabolic regulators in fungi, large factors with NF-6B-likebinding properties, cys6 cysteine-zinc cluster, zinc fingers ofalternating composition, homeo domain), Helix-turn-helix (homeo domainonly, POU domain factors, paired box, fork head/winged helix, heat shockfactors, tryptophan clusters, TEA domain), beta-Scaffold Factors withminor groove contacts (RHR, STAT, p53, MADS box, beta-Barrel alpha-helixtranscription factors, TATA-binding proteins, HMG, heteromeric CCAATfactors, grainyhead, cold-shock domain factors, runt), Othertranscription factors (copper fist proteins, HMGI(Y), Pocket domain, E1A-like factors, AP2/EREBP-related factors).

In addition to its primary structure, RNA has the ability to fold intocomplex tertiary structures consisting of such local motifs as loops,bulges, pseudoknots, and turns. It is not surprising that, when theyoccur in RNAs that interact with proteins, these local structures arefound to play important roles in protein-RNA interactions. Thisdiversity of local and tertiary structure, however, makes it impossibleto design synthetic agents with general, simple-to-use recognition rulesanalogous to those for the formation of double- and triple-helicalnucleic acids. Tripeptides have been identified that are able tospecifically bind to tertiary structures of nucleic acids (Hwang et al.,Proc. Natl. Acad. Sci. USA 96:12997 (1999)). Thus, in one embodiment,these tripeptides may be used as modulators of nucleic acid ligandactivity.

Peptide-based modulators of nucleic acid ligands represent analternative molecular class of modulators to oligonucleotides or theiranalogues. This class of modulators are particularly prove useful whensufficiently active oligonucleotide modulators of a target nucleic acidligand can not be isolated due to the lack of sufficient single-strandedregions to promote nucleation between the target and the oligonucleotidemodulator. In addition, peptide modulators provide differentbioavailabilities and pharmacokinetics than oligonucleotide modulators,and therefore, for some target nucleic acid ligands may be preferable.

Several strategies to isolate peptides capable of binding to and therebymodulating the activity of a target nucleic acid ligand are available.For example, encoded peptide combinatorial libraries immobilized onbeads have been described, and have been demonstrated to containpeptides able to bind viral RNA sequences and disrupt the interactionbetween the viral RNA and a viral regulatory protein that specificallybinds said RNA (Hwang et al., Proc Natl Acad Sci USA 96:12997 (1999)).Using such libraries, modulators of nucleic acid ligands can be isolatedby appending a label (eg. Dye) to the target nucleic acid ligand andincubating together the labeled-target and bead-immobilized peptidelibrary under conditions in which binding between some members of thelibrary and the nucleic acid are favored. The binding of the nucleicacid ligand to the specific peptide on a given bead causes the bead tobe “colored” by the label on the nucleic acid ligand, and thus enablesthe identification of peptides able to bind the target but simpleisolation of the bead. The direct interaction between peptides isolatedby such screening methods and the target nucleic acid ligand can beconfirmed and quantified using any number of the binding assaysdescribed to identify modulators of nucleic acid ligands. The ability ofsaid peptides to modulate the activity of the target nucleic acid ligandcan be confirmed in appropriate bioassays.

As another strategy, the target nucleic acid ligand can be immobilizedon a solid support (eg plastic, bead etc) by appending the appropriateaffinity handle (eg biotin for streptavidin coated surfaces) or reactivesite (eg. Aliphatic amine) to either end of the target nucleic acidligand during chemical synthesis. Alternatively, biotinylated RNA or RNAcontaining the desired modification can be prepared according tostandard transcription protocols, but including a 5-fold molar excess ofa 5′-biotin-modified GMP or otherwise modified GMP over GTP in thereaction mixture. Methods for synthesizing 5′-biotin-modified guanosinenucleotides and additional guanosine nucleotide derivatives andincorporating said GMP derivatives into in vitro transcripts aredescribed in WO 98/30720 entitled “Bioconjugation of Oligonucleotides”.Once the target RNA has been immobilized on a surface, peptides capableof binding said target RNA can be isolated by screening combinatorialpeptide libraries, for example, random phage display libraries forpeptides capable of binding the target nucleic acid ligand usingstandard methods for screening phage display libraries. Once isolated,the direct interaction between peptides and the target nucleic acidligand can be confirmed and quantified using any number of the bindingassays described to identify potential modulators of nucleic acidligands. The ability of said peptides to modulate the activity of thetarget nucleic acid ligand can be confirmed in appropriate bioassays.

In addition, the mass spectral assays described can be used to screenamino acid and small molecular weight peptide libraries to identifythose capable of binding to the target nucleic acid ligand (see U.S.Pat. Nos. 6,342,393; 6,329,146; 6,253,168; and 6,221,587). Onceidentified, the direct interaction between such agents and the targetnucleic acid ligand can be confirmed and quantified using any number ofthe binding assays described to identify potential modulators of nucleicacid ligands. The ability of said peptides to modulate the activity ofthe target nucleic acid ligand can be confirmed in appropriatebioassays.

In addition, USPs 5,834,184, 5,786,145 and 5,770,434 disclose methodsfor generating and screening for peptide modulators of the invention.

B. Oligosaccharides

Oligosaccharides can interact with nucleic acids. The antibioticaminoglycosides are products of Streptomyces species and are representedby streptomycin, kanamycin, tobramycin, neomycin, netilmicin, amikacinand gentamicin. These antibiotics exert their activity by binding tobacterial ribosomes and preventing the initiation of protein synthesis.Aminoglycoside antibiotics interact specifically with diverse RNAmolecules such as various ribozymes and the HIV-1's TAR and RREsequences. Aminoglycoside antibiotics that bind to the 30s ribosomalA-site RNA cause misreading of the genetic code and inhibittranslocation. The aminoglycoside antibiotic paromomycin bindsspecifically to the RNA oligonucleotide at the 30S A site. Thisantibiotic binds the major groove on the A-site RNA within a pocketcreated by an A-A pair and a single bulged adenine. There are severalinteractions that occur between the aminoglycoside chemical groups andthe conserved nucleotides in the RNA.

Thus, oligosaccharides, like aminoglycosides, can bind to nucleic acidsand can be used to modulate the activity of nucleic acid ligands. Inaddition, the mass spectral assays described can be used to screen foroligosaccharides (e.g., aminoglycosides), alone or in a mixture, thatare capable of binding to the target nucleic acid ligand (see U.S. Pat.Nos. 6,342,393; 6,329,146; 6,253,168; and 6,221,587).

D. Small Molecules

A small molecule that intercalates between the nucleic acid ligand andthe target or otherwise disrupts or modifies the binding between thenucleic acid ligand and target can also be used as the therapeuticregulator.

Such small molecules can be identified by screening candidates in anassay that measures binding changes between the nucleic acid ligand andthe target with and without the small molecule, or by using an in vivoor in vitro assay that measures the difference in biological effect ofthe nucleic acid ligand for the target with and without the smallmolecule. Once a small molecule is identified that exhibits the desiredeffect, techniques such as combinatorial approaches can be used tooptimize the chemical structure for the desired regulatory effect.

Assays suitable for use in screening small molecules are described inU.S. Pat. No. 5,834,199 and in further detail below. In addition, themass spectral assays described can be used to screen small moleculelibraries to identify those capable of binding to the target nucleicacid ligand (see U.S. Pat. Nos. 6,342,393; 6,329,146; 6,253,168; and6,221,587).

E. Chimeras, Fusion Products and Otherwise Linked Materials

The oligonucleotides or analogues thereof (including ribozymes orDNAzymes or peptide nucleic acids (PNAs) or mopholino nucleic acids(MNAs)), nucleic acid binding peptides, polypeptides or proteins(including nucleic acid binding tripeptides, oligosaccharides, and smallmolecules can be combined covalently or noncovalently to provide acombination regulator as desired. In one example, an oligonucleotide,PNA or MNA can be linked to a peptide or protein to provide desiredproperties or therapeutic effect. Alternatively, an oligonucleotide oranalogue thereof or peptide or protein can be linked to a small moleculeto enhance its properties. These materials can be directly linked orlinked via a spacer group as well known to those skilled in the art. Inone embodiment, the small molecule includes a radioligand or otherdetectable agent that can be monitored using standard techniques. Inthis way, the progress and location of the regulator can be monitored.Alternatively, the small molecule is a therapeutic that is brought tothe site of therapy by the regulator, and then optionally cleaved toprovide its therapeutic effect at the appropriate location. In a thirdembodiment, the small molecule is a chelator that joins two biologicalmaterials together to produce a combined effect.

The chimeras, fusion products or otherwise multicomponent regulator canbe identified and evaluated for therapeutic efficacy as described indetail below.

III. Identification and Selection of the Modulator

Standard binding assays can be used to identify and select modulators ofthe invention. Nonlimiting examples are gel shift assays and BIACOREassays. That is, test modulators can be contacted with the nucleic acidligands to be targeted under test conditions or typical physiologicalconditions and a determination made as to whether the test modulator infact binds the nucleic acid ligand. Test modulators that are found tobind the nucleic acid ligand can then be analyzed in an appropriatebioassay (which will vary depending on the aptamer and its targetmolecule, for example coagulation tests) to determine if the testmodulator can affect the biological effect caused by the nucleic acidligand on its target molecule.

The Gel-Shift assay is a technique used to assess binding capability.For example, a DNA fragment containing the test sequence is firstincubated with the test protein or a mixture containing putative bindingproteins, and then separated on a gel by electrophoresis. If the DNAfragment is bound by protein, it will be larger in size and itsmigration will therefore be retarded relative to that of the freefragment. For example, one method for a electrophoretic gel mobilityshift assay can be (a) contacting in a mixture a nucleic acid bindingprotein with a non-radioactive or radioactive labeled nucleic acidmolecule comprising a molecular probe under suitable conditions topromote specific binding interactions between the protein and the probein forming a complex, wherein said probe is selected from the groupconsisting of dsDNA, ssDNA, and RNA; (b) electrophoresing the mixture;(c) transferring, using positive pressure blot transfer or capillarytransfer, the complex to a membrane, wherein the membrane is positivelycharged nylon; and (d) detecting the complex bound to the membrane bydetecting the non-radioactive or radioactive label in the complex.

The Biacore technology measures binding events on the sensor chipsurface, so that the interactant attached to the surface determines thespecificity of the analysis. Testing the specificity of an interactioninvolves simply analyzing whether different molecules can bind to theimmobilized interactant. Binding gives an immediate change in thesurface plasmon resonance (SPR) signal, so that it is directly apparentwhether an interaction takes place or not. SPR-based biosensors monitorinteractions by measuring the mass concentration of biomolecules closeto a surface. The surface is made specific by attaching one of theinteracting partners. Sample containing the other partner(s) flows overthe surface: when molecules from the sample bind to the interactantattached to the surface, the local concentration changes and an SPRresponse is measured. The response is directly proportional to the massof molecules that bind to the surface.

SPR arises when light is reflected under certain conditions from aconducting film at the interface between two media of differentrefractive index. In the Biacore technology, the media are the sampleand the glass of the sensor chip, and the conducting film is a thinlayer of gold on the chip surface. SPR causes a reduction in theintensity of reflected light at a specific angle of reflection. Thisangle varies with the refractive index close to the surface on the sideopposite from the reflected light. When molecules in the sample bind tothe sensor surface, the concentration and therefore the refractive indexat the surface changes and an SPR response is detected. Plotting theresponse against time during the course of an interaction provides aquantitative measure of the progress of the interaction. The Biacoretechnology measures the angle of minimum reflected light intensity. Thelight is not absorbed by the sample: instead the light energy isdissipated through SPR in the gold film. SPR response values areexpressed in resonance units (RU). One RU represents a change of 0.0001°in the angle of the intensity minimum. For most proteins, this isroughly equivalent to a change in concentration of about 1 pg/mm2 on thesensor surface. The exact conversion factor between RU and surfaceconcentration depends on properties of the sensor surface and the natureof the molecule responsible for the concentration change.

There are a number of other assays that can determine whether anoligonucleotide or analogue thereof, peptide, polypeptide,oligosaccharide or small molecule can bind to the nucleic acid ligand ina manner such that the interaction with the target is modified. Forexample, electrophoretic mobility shift assays (EMSAs), titrationcalorimetry, scintillation proximity assays, sedimentation equilibriumassays using analytical ultracentrifugation (see for eg.www.cores.utah.edu/interaction), fluorescence polarization assays,fluorescence anisotropy assays, fluorescence intensity assays,fluorescence resonance energy transfer (FRET) assays, nitrocellulosefilter binding assays, ELISAs, ELONAs (see, for example, U.S. Pat. No.5,789,163), RIAs, or equilibrium dialysis assays can be used to evaluatethe ability of an agent to bind to a nucleic acid ligand. Direct assaysin which the interaction between the agent and the nucleic acid ligandis directly determined can be performed, or competition or displacementassays in which the ability of the agent to displace the nucleic acidligand from its target can be performed (for example, see Green, Belland Janjic, Biotechniques 30(5), 2001, p 1094 and U.S. Pat. No.6,306,598). Once a candidate modulating agent is identified, its abilityto modulate the activity of a nucleic acid ligand for its target can beconfirmed in a bioassay. Alternatively, if an agent is identified thatcan modulate the interaction of a nucleic acid ligand with its target,such binding assays can be used to verify that the agent is interactingdirectly with the nucleic acid ligand and can measure the affinity ofsaid interaction.

In another embodiment, mass spectrometry can be used for theidentification of an regulator that binds to a nucleic acid ligand, thesite(s) of interaction between the regulator and the nucleic acidligand, and the relative binding affinity of agents for the nucleic acidligand (see for example U.S. Pat. No. 6,329,146, Crooke et al). Suchmass spectral methods can also be used for screening chemical mixturesor libraries, especially combinatorial libraries, for individualcompounds that bind to a selected target nucleic acid ligand that can beused in as modulators of the nucleic acid ligand. Furthermore, massspectral techniques can be used to screen multiple target nucleic acidligands simultaneously against, e.g. a combinatorial library ofcompounds. Moreover, mass spectral techniques can be used to identifyinteraction between a plurality of molecular species, especially “small”molecules and a molecular interaction site on a target nucleic acidligand.

In vivo or in vitro assays that evaluate the effectiveness of aregulator in modifying the interaction between a nucleic acid ligand anda target are specific for the disorder being treated. There are amplestandard assays for biological properties that are well known and can beused. Examples of biological assays are provided in the patents cited inthis application that describe certain nucleic acid ligands for specificapplications.

As a nonlimiting example, coagulation tests can be performed asbioassays in clinical laboratories. These tests are generally functionalend-point assays, in which a patient sample (plasma or whole blood) isincubated with exogenous reagents that activate the coagulation cascade,and the time until clot formation is measured. The clotting time of thepatient sample is then compared to the clotting time of pooled normalplasma or whole blood to provide a standard measurement of the patient'shemostatic status. As described below, such clotting assays are commonlyused as screening tests that evaluate the functioning of both thepatient's intrinsic and extrinsic coagulation systems.

The Activated Clotting Time Test (ACT) is a screening test thatresembles the activated partial thromboplastin time (APTT) test, but isperformed using fresh whole blood samples. ACT can be used to monitor apatient's coagulation status in connection with clinical procedures,such as those that involve the administration of high doses of heparin(e.g., CPB and PTCA).

The Activated Partial Thromboplastin Time Test (APTT) is a commoncentral laboratory test, APTT is used to evaluate the intrinsiccoagulation pathway, which includes factors I, II, V, VIII, IX, X, XI,and XII. The test is performed using a plasma sample, in which theintrinsic pathway is activated by the addition of phospholipid, anactivator (ellagic acid, kaolin, or micronized silica), and Ca²⁺.Formation of the Xase and prothrombinase complexes on the surface of thephospholipid enables prothrombin to be converted into thrombin, withsubsequent clot formation. The result of the APTT test is the time (inseconds) required for this reaction. APTT can be used to assess theoverall competence of a patient's coagulation system, as a preoperativescreening test for bleeding tendencies, and as a routine test formonitoring heparin therapy. Another example of the APTT is performed asfollows. First, blood plasma is collected after the blood was subjectedto centrifugal separation, and then acting is added thereto. Inaddition, calcium chloride is added. The period of time is measureduntil coagulation is formed More in detail, the blood plasma is storedin a refrigerator after it was obtained through centrifugal separationof blood. Activating agent of 0.1 ml, which was warmed in a water havinga temperature of 37° C. for a minute, is poured into a test tubecontaining 0.1 ml of the plasma The mixture is allowed to stay in awater of 37° C. for two minutes. Then to this mixture, 0.1 ml of CaCl₂of 0.02 M, which had been placed in a water having a temperature of 37°C., is added under pressure. At this moment a stop watch is switched on.The test tube is heated in the water of 37° C. for 25 seconds. The testtube is taken out, and if coagulation is observed, the stop watch isturned off. This is one way in which the blood coagulating time can bemeasured.

The bleeding time test can be used for the diagnosis of hemostaticdysfunction, von Willebrand's disease, and vascular disorders. It alsocan be used to screen for platelet abnormalities prior to surgery. Thetest is performed by making a small incision on the forearm and wickingaway the blood from the wound site. The time it takes for bleeding tostop is recorded and in control subjects is approximately 3.5 minutes.Prolongation of the bleeding time is indicative of qualitative orquantitative platelet defects.

The Prothrombin Time Test (PT), which was first described by Quick in1935, measures the tissue factor-induced coagulation time of blood orplasma. It is used as a screening test to evaluate the integrity of theextrinsic coagulation pathway, and is sensitive to coagulation factorsI, II, V, VII, and X. The test can be performed by adding thromboplastinand Ca²⁺ to a patient sample and measuring the time for clot formation.A prolonged clotting time suggests the presence of an inhibitor to, or adeficiency in, one or more of the coagulation factors of the extrinsicpathway. But PT clotting time can also be prolonged for patients onwarfarin therapy, or for those with vitamin K deficiency or liverdysfunction. The PT test can provide an assessment of the extrinsiccoagulation pathway, and is widely used to monitor oral anticoagulationtherapy.

The Thrombin Clotting Time Test (TCT) measures the rate of a patient'sclot formation compared to that of a normal plasma control. The test canbe performed by adding a standard amount of thrombin to a patient'splasma that has been depleted of platelets, and measuring the timerequired for a clot to form. This test has been used as an aid in thediagnosis of disseminated intravascular coagulation (DIC) and liverdisease.

There are also a number of tests that can be used in the diagnosis of apatient's coagulative status. These fall into two categories: complextests, some of which are based on the screening tests outlined above,and immunoassays. Complex Tests include specific factor assays based onlaboratory tests, such as the APTT, PT, and TCT tests. One assaymeasures the level of the activation peptide factor IXa or the factorIXa-antithrombin III complex. These measurements are used to determinethe levels of factor IXa or factor VII-tissue mediated complex. Assaysfor activated protein C resistance, antithrombin, protein C deficiency,and protein S deficiency are also part of this group. Asymptomaticindividuals who have heterogeneous deficiencies of proteins C and S, andresistance to activated protein C, have significantly elevated levels ofthe prothrombin fragment F1.2 compared to controls.

V. Method of Regulating Nucleic Acid Ligand Therapy

A method of modulating biological activity is provided that includes (i)administering to a patient in need thereof a nucleic acid ligand thatbinds to a target to produce a therapeutic effect, and then at theselected or desired time, (ii) administering to the patient a modulatorthat modifies the therapeutic effect. In one case, the modulator turnsoff the therapeutic effect. In another embodiment, the modulator reducesor minimizes but does not terminate the therapeutic effect. In yetanother embodiment, the modulator enhances the therapeutic effect.

The base therapeutic effect is determined by the target and the nucleicacid ligand. The modification of the therapeutic effect is determined bythe modulator. Any known or developed nucleic acid ligand can beregulated in accordance with this invention.

In one embodiment, the method comprises: (a) administering to a patient,including any warm-blooded vertebrate in need thereof, an effectiveamount of a nucleic acid ligand or DNA aptamer that selectively binds acoagulation pathway factor, the RNA aptamer having a dissociationconstant for the coagulation pathway factor of about 20 nM or less; (b)modulating the biological activity of the coagulation pathway factor inthe warm-blooded vertebrate through the administering of the RNA aptamerin step (a); and (c) providing an antidote to reverse the effects of theaptamer by administration of a modulator. For example, the modulators ofthe present invention can bind to nucleic acid ligands that targettissue factor (TF)/factor VIIa (FVIIa), factor VIIIa (FVIIIa)/factor IXa(FIXa), factor Va (FVa/factor Xa (Fxa) enzyme complexes and plateletreceptors such as gp IIbIIIa and gp IbIX and modulate the effects of thenucleic acid ligand. This invention also provides antidote control ofplatelet inhibitors, antithrombotics and fibrinolytics.

At least three clinical scenarios exist in which the ability to rapidlyreverse the activity of an antithrombotic or anticoagulant nucleic acidligand is desirable. The first case is when anticoagulant orantithrombotic treatment leads to hemorrhage, including intracranial orgastrointestinal hemorrhage. While identifying safer target proteins mayreduce this risk, the potential for morbidity or mortality from thistype of bleeding event is such that the risk can not be overlooked. Thesecond case is when emergency surgery is required for patients who havereceived antithrombotic treatment. This clinical situation arises in alow percentage of patients who require emergency coronary artery bypassgrafts while undergoing percutaneous coronary intervention under thecoverage of GPIIb/IIIa inhibitors. Current practice in this situation isto allow for clearance of the compound (for small molecule antagonistssuch as eptifibatide), which may take 2-4 hours, or platelet infusion(for Abciximab treatment). The third case is when an anticoagulantnucleic acid ligand is used during a cardiopulmonary bypass procedure.Bypass patients are predisposed to post operative bleeding. In eachcase, acute reversal of the anticoagulant effects of a compound via anantidote (e.g., an oligonucleotide modulator of the invention targetedto an anticoagulant or antithrombotic nucleic acid ligand) allows forimproved, and likely safer, medical control of the anticoagulant orantithrombotic compound.

A method of treating cardiovascular disease in patients is also providedin accordance with the present invention. The method comprisesadministering an effective amount of an RNA aptamer that selectivelybinds a coagulation pathway factor, the RNA aptamer having adissociation constant for the coagulation pathway factor of about 20 nMor less, to a vertebrate subject suffering from cardiovascular disease,whereby cardiovascular disease in the vertebrate subject is treated,then providing an antidote to reverse the effects of the aptamer byadministration of a modulator.

A method of modulating E2F activity in patient, including a warm-bloodedvertebrate, in which such modulation is desired is also provided. Themethod comprises: (a) administering to the warm-blooded vertebrate aneffective amount of an RNA aptamer that selectively binds an E2F familymember, the RNA aptamer having a dissociation constant for the E2Ffamily member of about 20 nM or less; (b) modulating E2F in thewarm-blooded vertebrate through the administering of the RNA aptamer ofstep (a); and (c) providing an antidote to reverse the effects of theaptamer by administration of a modulator.

The patient treated in the present invention in its many embodiments isdesirably a human patient, although it is to be understood that theprinciples of the invention indicate that the invention is effectivewith respect to all vertebrate species, including warm-blood vertebrates(e.g., birds and mammals), which are intended to be included in the term“patient”. In this context, a mammal is understood to include anymammalian species in which treatment of cardiovascular disease isdesirable, particularly agricultural and domestic mammalian species.

Contemplated is the treatment of mammals such as humans, as well asthose mammals of importance due to being endangered (such as Siberiantigers), of economical importance (animals raised on farms forconsumption by humans and/or social importance (animals kept as pets orin zoos) to humans, for instance, carnivores other than humans such ascats and dogs), swine (pigs, hogs, and wild board), ruminants (such ascattle, oxen, sheep, giraffes, deer goats, bison, and camels), andhorses. Also contemplated is the treatment of birds, including thetreatment of those kinds of birds that are endangered, kept in zoos, aswell as fowl, and more particularly domesticated fowl, i.e., poultry,such as turkeys, chickens, ducks, geese, guinea fowl, and the like, asthey are also of economical importance to humans.

The present method for treating cardiovascular disease in a tissuecontemplates contacting a tissue in which cardiovascular disease isoccurring, or is at risk for occurring, with a composition comprising atherapeutically effective amount of an RNA aptamer capable of binding acoagulation factor as well as providing an antidote to reverse theeffects of the aptamer by administration of a modulator. Thus, themethod comprises administering to a patient a therapeutically effectiveamount of a physiologically tolerable composition containing the RNAaptamer as well as a method to provide an antidote to reverse theeffects of the aptamer by administration of a modulator.

The dosage ranges for the administration of the modulator depend uponthe form of the modulator, and its potency, as described further herein,and are amounts large enough to produce the desired effect. Forsituations in which coagulation is modulated, which can correspondinglyameliorate cardiovascular disease and the symptoms of cardiovasculardisease, the dosage should not be so large as to cause adverse sideeffects, such as hyperviscosity syndromes, pulmonary edema, congestiveheart failure, and the like. Generally, the dosage will vary with theage, condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The individual physician in theevent of any complication can also adjust the dosage.

A therapeutically effective amount is an amount of a modulatorsufficient to produce a measurable modulation of the effects of thenucleic acid ligand, including but not limited to acoagulation-modulating amount, an E2F activity-modulating amountcoagulation, and/or angiogenesis factor activity (e.g., Ang1 or Ang2activity)-modulating amount. Modulation of coagulation, E2F activity,and/or angiogenesis factor activity (e.g., Ang1 or Ang2 activity) can bemeasured in situ by immunohistochemistry by methods disclosed in theExamples, or by other methods known to one skilled in the art.

A preferred modulator has the ability to substantially bind to a nucleicacid ligand in solution at modulator concentrations of less than one(1.) micromolar (μM), preferably less than 0.1 μM, and more preferablyless than 0.01 μM. By “substantially” is meant that at least a 50percent reduction in target biological activity is observed bymodulation in the presence of the a target, and at 50% reduction isreferred to herein as an IC₅₀ value.

Preferred modes of administration of the materials of the presentinvention to a mammalian host are parenteral, intravenous, intradermal,intra-articular, intra-synovial, intrathecal, intra-arterial,intracardiac, intramuscular, subcutaneous, intraorbital, intracapsular,intraspinal, intrasternal, topical, transdermal patch, via rectal,vaginal or urethral suppository, peritoneal, percutaneous, nasal spray,surgical implant, internal surgical paint, infusion pump or viacatheter. In one embodiment, the agent and carrier are administered in aslow release formulation such as an implant, bolus, microparticle,microsphere, nanoparticle or nanosphere. For standard information onpharmaceutical formulations, see Ansel, et al., Pharmaceutical DosageForms and Drug Delivery Systems, Sixth Edition, Williams & Wilkins(1995).

The modulators of the present invention can be preferably administeredparenterally by injection or by gradual infusion over time. Although thetissue to be treated can typically be accessed in the body by systemicadministration and therefore most often treated by intravenousadministration of therapeutic compositions, other tissues and deliverytechniques are provided where there is a likelihood that the tissuetargeted contains the target molecule. Thus, modulators of the presentinvention are typically administered orally, topically to a vasculartissue, intravenously, intraperitoneally, intramuscularly,subcutaneously, intra-cavity, transdermally, and can be delivered byperistaltic techniques. As noted above, the pharmaceutical compositionscan be provided to the individual by a variety of routes such orally,topically to a vascular tissue, intravenously, intraperitoneally,intramuscularly, subcutaneously, intra-cavity, transdermally, and can bedelivered by peristaltic techniques. Representative, non-limingapproaches for topical administration to a vascular tissue include (1)coating or impregnating a blood vessel tissue with a gel comprising anucleic acid ligand, for delivery in vivo, e.g., by implanting thecoated or impregnated vessel in place of a damaged or diseased vesseltissue segment that was removed or by-passed; (2) delivery via acatheter to a vessel in which delivery is desired; (3) pumping a nucleicacid ligand composition into a vessel that is to be implanted into apatient. Alternatively, the nucleic acid ligand can be introduced intocells by microinjection, or by liposome encapsulation. Advantageously,nucleic acid ligands of the present invention can be administered in asingle daily dose, or the total daily dosage can be administered inseveral divided doses. Thereafter, the modulator is provided by anysuitable means to alter the effect of the nucleic acid ligand byadministration of the modulator.

The therapeutic compositions comprising modulator polypeptides of thepresent invention are conventionally administered intravenously, as byinjection of a unit dose, for example. The term “unit dose” when used inreference to a therapeutic composition of the present invention refersto physically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

As used herein, the terms “pharmaceutically acceptable,”“physiologically tolerable,” and grammatical variations, thereof, asthey refer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration without substantial or debilitating toxic side effects.

Pharmaceutically useful compositions comprising a modulator of thepresent invention can be formulated according to known methods such asby the admixture of a pharmaceutically acceptable carrier. Examples ofsuch carriers and methods of formulation can be found in Remington 'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the aptamer. Such compositions cancontain admixtures of more than one modulator.

The effective amount can vary according to a variety of factors such asthe individual's condition, weight, sex, age and amount of nucleic acidlidgand administered. Other factors include the mode of administration.Generally, the compositions will be administered in dosages adjusted forbody weight, e.g., dosages ranging from about 1 μg/kg body weight toabout 100 mg/kg body weight, preferably, 1 mg/kg body weight to 50 mg/kgbody weight.

Modulators of nucleic acid ligands can be particularly useful for thetreatment of diseases where it is beneficial to inhibit coagulation, E2Factivity, and/or angiogenesis factor activity (e.g., Ang1 or Ang2activity), or prevent such activity from occurring. The pharmaceuticalcompositions are administered in therapeutically effective amounts, thatis, in amounts sufficient to generate a coagulation-, E2F activity-,and/or angiogenesis factor activity (e.g., Ang1 or Ang2activity)-modulating response, or in prophylactically effective amounts,that is in amounts sufficient to prevent a coagulation factor fromacting in a coagulation cascade, to prevent an E2F activity-mediatedresponse, or to prevent an angiogenesis factor activity (e.g., Ang1 orAng2 activity)-mediated response. The therapeutically effective amountand prophylactically effective amount can vary according to the type ofmodulator. The pharmaceutical composition can be administered in singleor multiple doses.

Generally, oligonucleotide modulators of the invention can beadministered using established protocols used in antisense therapies.The data presented in Example 6 indicate the activity of a therapeuticnucleic acid ligand can be modulated by the intravenous infusion ofantidote oligonucleotides into a human or other animal. Furthermore,because the modulator's activity is durable, once the desired level ofmodulation of the nucleic acid ligand by the modulator is achieved,infusion of the modulator can be terminated, allowing residual modulatorto clear the human or animal. This allows for subsequent re-treatmentwith the nucleic acid ligand as needed. Alternatively, and in view ofthe specificity of the modulators of the invention, subsequent treatmentcan involve the use of a second, different nucleic acid ligand/modulator(e.g., oligonucleotide) pair.

Modulators synthesized or identified according to the methods disclosedherein can be used alone at appropriate dosages defined by routinetesting in order to obtain optimal modulation of nucleic acid ligandactivity in coagulation, E2F, and/or angiogenesis factor cascades (e.g.,Ang1 or Ang2 activity) while minimizing any potential toxicity. Inaddition, co-administration or sequential administration of other agentscan be desirable. For combination treatment with more than one activeagent, where the active agents are in separate dosage formulations, theactive agents can be administered concurrently, or they each can beadministered at separately staggered times.

The dosage regimen utilizing the modulators of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal and hepatic function of the patient; and the particularmodulator employed. A physician of ordinary skill can readily determineand prescribe the effective amount of the aptamer required to prevent,counter or arrest the progress of the condition. Optimal precision inachieving concentrations of modulator within the range that yieldsefficacy without toxicity requires a regimen based on the kinetics ofthe modulator's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of themodulator.

In the methods of the present invention, the modulators herein describedin detail can form the active ingredient, and are typically administeredin admixture with suitable pharmaceutical diluents, excipients orcarriers (collectively referred to herein as “carrier” materials)suitably selected with respect to the intended form of administration,that is, oral tablets, capsules, elixirs, syrup, suppositories, gels andthe like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders includewithout limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methyl-cellulose and thelike. Other dispersing agents that can be employed include glycerin andthe like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations that generally containsuitable preservatives are employed when intravenous administration isdesired.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as,e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 mydstyl propionate, and the like, to form, e.g.,alcoholic solutions, topical cleansers, cleansing creams, skin gels,skin lotions, and shampoos in cream or gel formulations.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine or phosphatidylcholines.

The compounds of the present invention can also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropylmethacryl-amidephenol,polyhydroxy-ethylaspartamidepbenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention can be coupled (preferably via a covalent linkage) toa class of biodegradable polymers useful in achieving controlled releaseof a drug, for example, polyethylene glycol (PEG), polylacetic acid,polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked oramphipathic block copolymers of hydrogels. Cholesterol and similarmolecules can be linked to the aptamers to increase and prolongbioavailability.

The oligonucleotide modulators can be administered directly (e.g., aloneor in a liposomal formulation or complexed to a carrier (e.g., PEG))(see for example, U.S. Pat. No. 6,147,204, U.S. Pat. No. 6,011,020).

The nucleic acid ligand or modulator can be comprised of aoligonucleotide modulator attached to a non-immunogenic, high molecularweight compound such as polyethylene glycol (PEG). In this embodiment,the pharmacokinetic properties of the complex are improved relative tothe nucleic acid ligand alone. As discussed supra, the association couldbe through covalent bonds or non-covalent interactions. In oneembodiment, the modulator is associated with the PEG molecule throughcovalent bonds. Also, as discussed supra, where covalent attachment isemployed, PEG may be covalently bound to a variety of positions on theoligonucleotide modulator. In the preferred embodiment, anoligonucleotide modulator is bonded to the 5′ thiol through a maleimideor vinyl sulfone functionality. In one embodiment, a plurality ofmodulators can be associated with a single PEG molecule. The modulatorcan be to the same or different target. In embodiments where there aremultiple modulators to the same nucleic acid ligand, there is anincrease in avidity due to multiple binding interactions with theligand. In yet a further embodiment, a plurality of PEG molecules can beattached to each other. In this embodiment, one or more modulators tothe same target or different targets can be associated with each PEGmolecule. This also results in an increase in avidity of each modulatorto its target. In embodiments where multiple modulators specific for thesame target are attached to PEG, there is the possibility of bringingthe same targets in close proximity to each other in order to generatespecific interactions between the same targets. Where multiplemodulators specific for different targets are attached to PEG, there isthe possibility of bringing the distinct targets in close proximity toeach other in order to generate specific interactions between thetargets. In addition, in embodiments where there are modulators to thesame target or different targets associated with PEG, a drug can also beassociated with PEG. Thus the domplex would provide targeted delivery ofthe drug, with PEG serving as a Linker.

One problem encountered in the therapeutic and in vivo diagnostic use ofnucleic acids is that oligonucleotides in their phosphodiester form maybe quickly degraded in body fluids by intracellular and extracellularenzymes such as endonucleases and exonucleases before the desired effectis manifest. Certain chemical modifications of the nucleic acid can bemade to increase the in vivo stability of the nucleic acid or to enhanceor to mediate the delivery of the nucleic acid. Modifications of thenucleic acid ligands contemplated in this invention include, but are notlimited to, those which provide other chemical groups that incorporateadditional charge, polarizability, hydrophobicity, hydrogen bonding,electrostatic interaction, and fluxionality to the nucleic acid bases orto the nucleic acid as a whole. Such modifications include, but are notlimited to, 2′-position sugar modifications, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atexocyclic amines, substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil; backbone modifications, phosphorothioate or alkylphosphate modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.

Lipophilic compounds and non-immunogenic high molecular weight compoundswith which the modulators of the invention can be formulated for use inthe present invention and can be prepared by any of the varioustechniques presently known in the art or subsequently developed.Typically, they are prepared from a phospholipid, for example,distearoyl phosphatidylcholine, and may include other materials such asneutral lipids, for example, cholesterol, and also surface modifierssuch as positively charged (e.g., sterylamine or aminomannose oraminomannitol derivatives of cholesterol) or negatively charged (e.g.,diacetyl phosphate, phosphatidyl glycerol) compounds. Multilamellarliposomes can be formed by the conventional technique, that is, bydepositing a selected lipid on the inside wall of a suitable containeror vessel by dissolving the lipid in an appropriate solvent, and thenevaporating the solvent to leave a thin film on the inside of the vesselor by spray drying. An aqueous phase is then added to the vessel with aswirling or vortexing motion which results in the formation of MLVs. UVscan then be formed by homogenization, sonication or extrusion (throughfilters) of MLV's. In addition, UVs can be formed by detergent removaltechniques. In certain embodiments of this invention, the complexcomprises a liposome with a targeting nucleic acid ligand(s) associatedwith the surface of the liposome and an encapsulated therapeutic ordiagnostic agent. Preformed liposomes can be modified to associate withthe nucleic acid ligands. For example, a cationic liposome associatesthrough electrostatic interactions with the nucleic acid. Alternatively,a nucleic acid attached to a lipophilic compound, such as cholesterol,can be added to preformed liposomes whereby the cholesterol becomesassociated with the liposomal membrane. Alternatively, the nucleic acidcan be associated with the liposome during the formulation of theliposome. Preferably, the nucleic acid is associated with the liposomeby loading into preformed liposomes.

Alternatively, oligonucleotide modulators of the invention can beproduced in vivo following administration of a construct comprising asequence encoding the oligonucleotide. Techniques available foreffecting intracellular delivery of RNA modulators of gene expressioncan be used (see generally Sullenger et al., Mol. Cell. Biol. 10:6512(1990)).

Certain aspects of the invention can be described in greater detail inthe non-limiting Examples that follow.

EXAMPLE 1 Aptamer to Factor IXa

Nuclease-resistant 2′-fluoro pyrimidine-modified aptamers to humancoagulation Factor IXa were generated as described in WO 0226932 A2.Eight iterative cycles of selection were performed, yielding a family of16 aptamers with high affinity for FIXa; K_(D)'s ranging from ˜0.6-15 nMin physiologic salt and pH at 37° C. Comparative sequence analysis madeit possible to predict and synthesize the minimized version of thehighest affinity aptamer, termed RNA 9.3t (“t” for truncate), shown inFIG. 1. This 34 nucleotide aptamer has a molecular weight of 11.5 kDaand binds FIXa with essentially the same affinity as the full-lengthsequence (K_(D)0.6 nM). As a control, a mutant version of RNA 9.3ttermed 9.3tM was synthesized in which the absolutely conserved A's inthe internal loop were mutated to G's (FIG. 1). This aptamer binds FIXawith a K_(D)>5 μM as determined by competition binding assays. In allactivity assays, RNA 9.3tM is employed as a control to measure anynon-specific effects caused by an aptamer of this composition. Aptamer9.3t blocks FX activation by FVIIIa/FIXa/lipids, and also partiallyblocks synthetic substrate hydrolysis by FIXa.

To examine the specificity of the aptamer for FIXa versus structurallysimilar coagulation factors, the affinity of 9.3t for FIX, FVIIa, FXa,FXIa and APC was measured in direct binding assays as previouslydescribed (Rusconi et al., Thrombosis and Haemostasis 83:841-848(2000)). The aptamer binds FIX only ˜5-50 fold less tightly than FIXa.It failed to exhibit significant binding at protein concentrations up to5 μM (fraction RNA bound<10%) to any of the other proteins tested.Therefore, the specificity of aptamer 9.3t for FIXa versus FVIIa, FXa,FXIa or APC is >5000 fold.

To determine the anticoagulant potency of RNA 9.3t, the ability of 9.3tto prolong the clotting time of human plasma has been evaluated inactivated partial thromboplastin time (APTT) and prothrombin time (PT)clotting assays. Aptamer 9.3t, but not control aptamer 9.3tM, was ableto prolong the clotting time of human plasma in a dose dependent manner(FIG. 2). Neither aptamer had an effect on the PT, demonstrating thefunctional specificity of aptamer 9.3t for FIXa and demonstrating thatthis type of oligonucleotide, at concentrations up to 1 μM, does notnon-specifically increase the clotting time of human plasma. Thus, RNA9.3 is a potent anticoagulant, with a maximal effect on the APTT similarto that observed in FIX deficient plasma. Similar experiments have beenperformed in porcine plasma, and similar potency and specificity havebeen observed.

To determine if this molecule is capable of inhibiting FIX/FIXa activityin vivo, the ability of this aptamer to systemically anticoagulate small(1.5-4 kg) pigs following bolus intravenous injection was tested. Forthese experiments, an intravenous catheter was placed in the femoralvein for sample injection and an arterial catheter was placed in afemoral artery of the animal for serial withdrawal of blood samples. Apre-injection blood sample was taken, and the ACT time measured on siteto establish a baseline whole blood clot time for the animal. Aptamer9.3t at doses of 0.5 mg/kg (n=4) and 1.0 mg/kg (n=4), aptamer 9.3tM at1.0 mg/kg (n=3) or vehicle (n=3) was then delivered by intravenous bolusinjection. Blood samples were taken at various times post injection, andthe ACT immediately determined. Additional blood was drawn fordetermination of APTT's at different times post-injection. As shown inFIG. 3, aptamer 9.3t, but not the control aptamer 9.3tM or vehicle, wasable to inhibit FIXa activity in vivo as evidenced by a significantdose-dependent increase in the animal's ACT post injection. As shown inFIG. 4, aptamer 9.3 specifically prolonged the APTT, but not PT of theanimals in a dose dependent manner. Using the in vitro dose responsecurves from the APTT experiments, the change in 9.3t plasmaconcentration over time following injection can be estimated.

As an attempt to increase the bioavailability of 9.3t, a version of thisaptamer with a 5′cholesterol moiety was synthesized, termed 9.3t-C. Thecholesterol modified aptamer retained high affinity binding to FIXa(FIG. 5A) and potent anticoagulant activity (FIGS. 5B and 5C). The invivo effects of this modification on the circulating half-life of 9.3t-Cversus 9.3t (n=2 animal for each aptamer) have been tested using the pigsystemic anticoagulation model. Following injection of 0.5 mg/kg 9.3t-C,the ACT of the animal increased ˜1.4 fold and was sustained at thislevel for 1 hour post injection (FIG. 6A). FIG. 6B shows analysis of theAPTT and PT of the animals from this experiment. While the anticoagulantpotency of the two aptamers is similar, the duration of theanticoagulant effect of 9.3t-C is significantly longer (FIG. 6C).

EXAMPLE 2 Oligonucleotide that Reverses Interaction of the FIXa Aptamerwith Coagulation FIXa

The secondary structure model of 9.3t is shown in FIG. 1 and wasdeveloped from comparative sequence analysis of the related FIXa aptamersequences shown in FIG. 7. These data strongly support formation of thestem-looped structure depicted in FIG. 1. In addition, mutationalanalysis of aptamer 9.3t demonstrated that disruption of either stem 1or stem 2 resulted in a greater than 1000 fold loss of affinity forFIXa. Therefore, a 17 residue all 2′Omethyl oligonucleotide was designed(sequence 5′ auggggaggcagcauua 3′) (Anti-D1) complementary to the 3′halfof aptamer 9.3t beginning at the 5′ end of loop 2 (L2 in FIG. 7) andextending to the 3′ end of the aptamer. This design allows fornucleation of an intermolecular duplex between the oligonucleotide andloop 2 of the aptamer. Also, formation of the intermolecular duplex isthermodynamically favored due to both the length and base composition ofthe complimentary oligonucleotide. A ribonucleotide duplex of thissequence has calculated free energy of −26.03 kcal/mol and a predictedT_(m) of 75.4° C. The half-life of such a duplex at 37° C. would greatlyexceed 24 hours.

To determine if this oligonucleotide could denature aptamer 9.3t,radiolabeled aptamer 9.3t (125 nM) was incubated with increasingconcentrations of the “antidote” oligonucleotide (from equimolar to an 8fold molar excess) at 37° C. for 15 minutes (-heat FIG. 8), and theamount of intermolecular duplex formed was visualized by native gelelectrophoresis (12% acrylamide, 150 mM NaCl, 2 mM CaCl₂, run in1Xtris-borate buffer +2 mM CaCl₂) followed by phosphorimaging (FIG. 8).To generate an oligonucleotide-aptamer complex as a gel-mobilitycontrol, the oligonucleotide was annealed to aptamer 9.3t by heating theaptamer and an 8 fold molar excess of the oligonucleotide at 95° C. for5 minutes prior to the 37° C. incubation (+heat FIG. 8). The same set ofexperiments was performed with a nonsense oligonucleotide of the samebase composition as the antidote oligonucleotide (N.S. in FIG. 8). Ascan be seen in FIG. 8, the antidote oligonucleotide readily denaturesaptamer 9.3t as evidenced by near complete formation of theoligonucleotide-aptamer complex when the oligonucleotide was present atan 8 fold molar excess to the aptamer. In addition, this interaction isvery specific, as no complex is observed, with or without heating,between the aptamer and the nonsense control oligonucleotide.

To determine if this antidote oligonucleotide could reverse theanticoagulant activity of aptamer 9.3t, the APTT of pooled human plasmawas measured in the presence of 50 nM 9.3t and increasing concentrationsof the antidote oligonucleotide (FIGS. 9A and 9B). In this experiment,aptamer 9.3t was pre-incubated 5 minutes in plasma prior to addition ofthe antidote oligonucleotide to generate aptamer-FIX complexes, theantidote or nonsense oligonucleotide were then added, and the incubationcontinued for an additional 10 minutes prior to adding CaCl₂ to initiateclot formation. As shown in FIGS. 9A and 9B, the antidoteoligonucleotide is able to effectively reverse about 80% of theanticoagulant activity of aptamer 9.3t. However, the molar excess ofantidote oligonucleotide required to achieve this effect issubstantially larger than the amount required to effectively denaturethe aptamer in the absence of protein.

EXAMPLE 3 Specificity of Oligonucleotide Antidotes

In order to assess the specificity of an oligonucleotide antidote,aptamer 9.20t was prepared (see FIG. 10A). Stem 1 of 9.20t is identicalto stem 1 of aptamer 9.3t, as is the 3′ half of stem 2. The maindifferences between 9.20t and 9.3t are found in loop 2 and loop 3(compare FIG. 1 with FIG. 10A).

Aptamer 9.20t binds FIXa with a K_(D) comparable to that of 9.3t. APTTassays were used to measure the clot time of human plasma as a functionof the concentration of aptamer 9.20t. Its in vitro anticoagulantpotency in human plasma was comparable to that of 9.3t (FIG. 10B).Aptamer 9.20t was added to human plasma at a concentration of 50 nM andallowed to bind to plasma FIX for 5 minutes. Varying concentrations ofantidote oligonucleotide Anti D1 were then added, and the APTT wasmeasured at 10 minutes after antidote addition to plasma. The relativechange in clot time caused by 9.20t addition to plasma was unaffected bythe addition of this antidote oligonucleotide complimentary to aptamer9.3t (FIG. 10C).

EXAMPLE 4 Tailed Aptamer 9.3t

In order to determine if a single-stranded “tail” added to the end of anaptamer promotes association of an “antidote” oligonucleotide with theaptamer, a 3′ tail was added to aptamer 9.3t, the tailed aptamer beingdesignated 9.3t-3NT (FIG. 11). The 3′ tail of 9.3t-3NT is a 3 nucleotide2′Omethyl modified RNA tail. This tail sequence was chosen to reduce thelikelihood of impacting the activity of the aptamer, and to reducepotential secondary structures within the complimentary antidoteoligonucleotide.

The affinity of aptamer 9.3t-3NT was compared to 9.3t in competitionbinding assays (FIG. 12A). The affinity of aptamer 9.3t-3NT for FIXa wascomparable to that of 9.3t (K_(D) 1.5 nM or less). APTT assays were usedto measure the clot time of human plasma as a function of theconcentration of aptamers 9.3t-3NT and 9.3t (FIG. 12B). Theanticoagulant activities of the aptamers were similar, and both aptamerswere able to completely inhibit FIX activity in human plasma.

Aptamer 9.3t-3NT was added to human plasma at a concentration of 50 nM(˜3 fold increase in APTT) and allowed to bind to plasma FIX for 5minutes. Varying concentrations of antidote oligonucleotides (see FIG.11) were then added, and the APTT was measured at 10 minutes afterantidote addition to plasma (FIG. 13A). The fraction of theanticoagulant activity reversed by the antidote oligonucleotide is thedifference between the APTT in the presence of aptamer alone and theAPTT in the presence of aptamer +antidote divided by the change in APTTover baseline in the presence of the aptamer alone (0=no effect,1=complete reversal). Each of the complimentary antidoteoligonucleotides tested was able to reverse>90% of the anticoagulantactivity of aptamer 9.3t-3NT within 10 minutes of addition in humanplasma, with AS3NT-3 demonstrating the most potent reversal activity(FIG. 13B). This reversal activity is comparable to the ability ofprotamine to reverse the APTT increase following heparin addition tohuman plasma.

The reversal of the anticoagulant activity of 9.3t-3NT by antidoteoligonucleotide AS3NT-3 was compared to the reversal of theanticoagulant activity of 9.3t by antidote oligonucleotide Anti D1. Theaddition of the tail to the aptamer increases the efficiency of reversalby an antidote oligonucleotide with sequences complimentary to the 3′tail of the aptamer (FIGS. 14A and 14B).

In addition to the foregoing, the following oligonucleotide modulatorshave been produced that target aptamer 9.3t, and are effective atreversing its anticoagulant activity in human plasma in vitro (all are2′Omethyl oligonucleotides):

Anti D T1: 5′ cau ggg gag gca gca uua 3′

AS 9.3t-2: 5′ cau ggg gag gca gca 3′

AS 9.3t-3: 5′ cau ggg gag gca 3′.

The following oligonucleotide modulators target aptamers 9.3t and9.3t-3NT, and are effective at reversing its anticoagulant activity inhuman plasma in vitro (all are 2′Omethyl oligonucleotides):

AS 5-1: 5′ gca uua cgc ggu aua guc ccc ua 3′

AS 5-2: 5′ cgc ggu aua guc ccc ua 3′.

The following oligonucleotide modulators target either aptamer 9.3t oraptamer 9.3t-3NT, and are mutant oligonucleotides designed to testspecific aspects of the design of modulating oligonucleotides to theseaptamers (all are 2′Omethyl oligonucleotides):

AS 9.3t-M: 5′ cau ggg gaa gca 3′ (SEQ ID NO:37)

AS 9.3t-3NOH: 5′ aug ggg agg ca 3′ (SEQ ID NO:38).

AS 3NT-3M: 5′ gac aug ggg aag ca 3′ (SEQ ID NO:39)

AS 3NT-3 MT: 5′ aca aug ggg agg ca 3′ (SEQ ID NO:40)

EXAMPLE 5 Antidote Oligonucleotide to Aptamer 9.3t

The antidote oligonucleotide 5-2C (5′CGC GGU AUA GUC CCC AU) but not ascrambled version of this antidote oligonucleotide, 5-2C scr, has beenshown to effectively reverse the activity of aptamers 9.3t and Peg-9.3tin vitro in human plasma. Peg-9.3t is 9.3t with a 40 KDa polyethyleneglycol appended to its 5′ end via a linker.

In these experiments, aptamer was added to plasma (50 nM 9.3t, 125 nMPeg-9.3t) and allowed to incubate for 5 minutes. Antidoteoligonucleotides were then added, and APTT assays were initiated 10minutes after antidote addition. The results are shown in FIG. 15.

EXAMPLE 6 Rapidity and Durability of Control of Aptamer 9.3t by AntidoteOligonucleotide

Aptamers 9.3t or Peg-9.3t were added to human plasma in vitro at a finalconcentration of 50 nM for 9.3t or 125 nM for Peg-9.3t, and allowed toincubate for 5 minutes at 37° C. Antidote oligonucleotide 5-2C at theindicated molar excess to the aptamer was then added, and the residualaptamer activity was determined by measuring the clotting time in APTTassays at the times indicated following antidote addition. The %residual anticoagulant activity equals 1—the ratio of(T_(Aptamer)alone−T_(Aptamer)+antidote) to(T_(Aptamer)alone−T_(baseline))×100, where T=APTT clot time.

The duration of the inactivation of the anticoagulant activity ofPeg-9.3t by antidote oligonucleotide 5-2C was measured in vitro in humanplasma. Briefly, Peg-9.3t was added to human plasma to a finalconcentration of 125 nM and allowed to incubate for 5 minutes. Antidoteoligonucleotide 5-2C was then added at a 10 fold molar excess, or in aparallel experiment buffer alone was added in place of the antidoteoligonucleotide, and the clotting time was measure in an APTT assays atvarious time points following antidote addition. The % residualanticoagulant activity was determined as above. The APTT of untreatedhuman plasma was also measured in parallel to establish a baselineclotting time at each time point. It was found that after 5 hours ofincubation at 37° C., the APTT of the untreated plasma began toincrease, indicating the loss of the clot forming activity of theplasma, and the experiment was thus stopped at 5 hours.

The data presented in FIGS. 16 and 17 demonstrate to the ability torapidly and durably control the anticoagulant activity of the FIXaantagonist aptamer 9.3t, and its derivatives, using antidoteoligonucleotides. Together these data demonstrate that the onset ofaction of the antidote is rapid, that the time needed for the antidoteto act is at least in part dependent on the antidote concentration, andthat once the antidote has inactivated the aptamer, this effect isdurable.

EXAMPLE 7 Antidote Oligonucleotide to Aptamer Against Coagulation FactorXa

Depicted in FIG. 18A is an aptamer (designated 11F7t) againstcoagulation factor Xa that is a potent anticoagulant in vitro in humanplasma. Depicted in FIG. 18B is a mutant version of aptamer 11F7t,designated 11F7t M. Alterations of the identity of the positions shownin FIG. 18B leads to a >1300 fold loss of affinity of the mutant aptamerfor coagulation Fxa. Varying concentrations of aptamers 11F7t and 11F7tM were added to human plasma in vitro, and the clot time was thenmeasured in a PT (FIG. 19A) or APTT assay (FIG. 19B). In FIG. 19, dottedlines indicate relative change in clot time of plasmas containing 10% orless than 1% the normal plasma level of FX, demonstrating the potentanticoagulant effects of aptamer 11F7t. All data are normalized to thebaseline for that day, so that a value of 1=no change in clot time.Aptamer 11F7t is also a potent anticoagulant of human plasma when assaysin PT clotting assays, as would be expected for a FXa inhibitor. Themutant aptamer, 11F7tM showed no anti coagulation activity in either thePT or APTT assay.

The following antidote oligonucleotides were screened for the ability toreverse the anticoagulant activity of aptamer 11F7t in vitro in humanplasma:

AO 5-1: 5′CUC GCU GGG GCU CUC 3′

AO 5-2: 5′UAU UAU CUC GCU GGG 3′

AO 3-1: 5′ AAG AGC GGG GCC AAG 3′

AO 3-3: 5′ GGG CCA AGU AUU AU 3′.

FIG. 20 shows the sequences of 11F7t to which these antidoteoligonucleotides are complimentary. As shown in FIG. 21A, the antidoteoligonucleotides effectively reverse the activity of aptamer 11F7t inhuman plasma. In these experiments, aptamer was added to plasma (finalconcentration 125 nM) and allowed to incubate for 5 minutes. Antidoteoligonucleotides were then added, and APTT assays were initiated 10minutes after antidote addition. In addition to the antidoteoligonucleotides described above, the following sequences were alsofound to have antidote activity against aptamer 11F7t:

AO 3-2: 5′CAA GAG CGG GGC CAA G 3′

AO 5-3: 5′CGA GUA WUA UCU UG 3′.

FIG. 21B shows the characterization of antidote 5-2 activity over alarger concentration range of antidote 5-2, and comparison to theantidote activity of a scrambled sequence version of antidote 5-2,5-2scr. The data demonstrate potent reversal activity of antidote 5-2,and specificity of antidote oligonucleotide activity as demonstrated bylack of reversal activity of AO 5-2scr.

FIGS. 22 and 23 relate to the ability to rapidly and durably control theanticoagulant activity of the FXa antagonist aptamer 11F7t, and itsderivatives, using antidote oligonucleotides. Together these datademonstrate that the onset of action of the antidote is rapid (FIG. 22),that the time needed for the antidote to act is at least in partdependent on the antidote concentration (FIG. 22), and that once theantidote has inactivated the aptamer, this effect is durable (FIG. 23).Together these data indicate that antidote oligonucleotides can beintravenously infused into a human or other animal would be a potentialmethod to use antidote oligonucleotides to modulate the activity of atherapeutic aptamer. Furthermore, because the antidote activity isdurable, once the desired level of modulation of the aptamer by theantidote is achieved, infusion of the antidote can be terminated,allowing residual antidote to clear the human or animal. This allows forsubsequent re-treatment of the human or animal with the aptamer asneeded.

EXAMPLE 8 Independent Functioning of Aptamer Antidote Pairs

To demonstrate that aptamer-antidote pairs (aptamer 9.3t and itsantidote A05-2c and aptamer 11F7t and its antidote A05-2) functionindependently of each other, aptamers were added to human plasma at 37°C., as indicated in FIG. 24, and allowed to incubate for 5 minutes.Antidotes were then added and the clotting activity was measured 10minutes after antidote addition in APTT assays. In all assays, bufferalone was substituted for aptamer or antidote in cases in which only oneaptamer or antidote was added to the plasma. All data was normalized tothe baseline for that day, so that a value of 1=no change in clot time.

Comparing sample Apt 1&2+AD1 to Apt 1 alone and sample Apt 1&2+AD2 toApt 2 alone (see FIG. 24), it is clear that antidote reversal activityis specific for the targeted aptamer (e.g., there was no loss of Apt 2activity in the presence of AD 1 and vice versa), and the activity ofthe antidote was effectively unchanged by the presence of the secondaptamer.

These results have two important implications. First, they demonstratethe ability to dose a patient with nucleic acid ligand 1 (e.g., 9.3t),reverse that nucleic acid ligand with its matched antidote, and thenre-treat the patient with a second nucleic acid ligand. Second, theresults demonstrate the utility of nucleic acid ligand-antidote pairsfor target validation, and the study of biochemical pathways. Theantidote enables one to determine that the response observed afterinhibiting a target protein with an nucleic acid ligand is due tospecifically inhibiting that protein. In addition, the antidote makes itpossible to determine if binding of the nucleic acid ligand to thetarget protein leads to protein turnover. For example, if upon antidoteaddition complete protein activity is restored, it argues that there wasno net change in protein concentration as a result of nucleic acidligand binding.

EXAMPLE 9 Aptamer PEG-9.3t and Antidote 5-2C Function in Plasma fromPatients with Heparin-Induced Thrombocytopenia (HIT)

The ability to control the anticoagulant activity of heparin withprotamine enables safer treatment of patients undergoing proceduresrequiring a high level of anticoagulation, in whom the post-proceduralrisk of hemorrhage is high. However, 3-5% of patients receiving heparindevelop a drug-induced immunologic response termed heparin-inducedthrombocytopenia (HIT), which contraindicates further treatment of thesepatients with heparin (Warkentin et al., Thromb Haemost 79:1-7. (1998)).This disorder is characterized by a decrease in the platelet count andan increased risk for new or recurrent life and limb-threateningthromboembolism (Warkentin et al., Thromb Haemost 79:1-7. (1998))Several alternative anticoagulants are available, but none of theseanticoagulants can be controlled by a reversing agent. Thissignificantly limits the treatment options for patients with HIT, andhemorrhagic complications and recurrent thromboembolism while undergoingtreatment are common Greinacher et al., Circulation 99:73-80. (1999),Lewis et al., Circulation 103:1838-1843. (2001)). Therefore, the abilityof aptamer Peg-9.3t and antidote 5-2C to serve as ananticoagulant-antidote pair in plasma samples from six patients with HITwas investigated, three with end-stage renal disease requiringhemodialysis and thus repeated anticoagulation, and three withthromboembolic complications requiring anticoagulant therapy. (Serologiccriteria included a positive heparin-induced platelet aggregation assay(Ortel et al., Thromb Haemost 67:292-296. (1992)) and/or elevatedheparin/platelet factor 4 antibody levels detected by ELISA (GTI, Inc.,Brookfield, Wis.). Five patients met both clinical and serologiccriteria; one patient fulfilled clinical criteria but had negativeserologic studies.) Aptamer PEG-9.3t prolonged the APTT clotting timesof plasma from all six patients, and antidote 5-2 was able toeffectively reverse this anticoagulant activity to the pre-treatmentbaseline of each patient (FIG. 25). Importantly, two patients werereceiving anticoagulant therapy at the time samples were taken (patient3 on danaproid sodium and patient 6 on warfarin), and PEG-9.3t additionto plasma from these patients increased the clot time over the treatmentbaseline and antidote 5-2C reversed this response back to the treatmentbaseline, demonstrating that in patient plasma this drug-antidote paircan function independently of an “on board” anticoagulant. In addition,treatment of these patient plasma samples with the control aptamer 9.3tMand antidote 5-2C yielded no increase in the clot time, furtherindicating that oligonucleotides of the composition of the aptamer orantidote do not inherently possess significant anticoagulant activity.

The ability of aptamer 11F7t and it's corresponding antidote 5-2 toserve as an anticoagulant-antidote pair in plasma samples from 2patients with HIT, one with end-stage renal disease requiringhemodialysis and thus repeated anticoagulation, and one withthromboembolic complications requiring anticoagulant therapy. Aptamer11F7t prolonged the APTT clotting times of plasma from both patients,and antidote 5-2 was able to effectively reverse this anticoagulantactivity to the pre-treatment baseline of each patient (FIG. 26).Importantly, these two patients were receiving anticoagulant therapy atthe time samples were taken (patient 3 on danaproid sodium and patient 6on warfarin), and 11F7t addition to plasma from these patients increasedthe clot time over the treatment baseline and antidote 5-2 reversed thisresponse back to the treatment baseline, demonstrating that in patientplasma this drug-antidote pair can function independently of an “onboard” anticoagulant. In addition, treatment of these patient plasmasamples with the control aptamer 9.3tM and antidote 5-2 yielded noincrease in the clot time, further indicating that oligonucleotides ofthe composition of the aptamer or antidote do not inherently possesssignificant anticoagulant activity.

All documents cited above are hereby incorporated in their entirety byreference.

1. A method of altering the affinity of an aptamer for a target moleculein a host comprising administering to a host a modulator that binds tothe aptamer, wherein conditions are such that the modulator binds to theaptamer and alters the affinity of the aptamer for the target molecule.2. The method according to claim 1 wherein upon binding of the modulatorto the aptamer, the affinity of the aptamer for the target molecule isreduced.
 3. The method according to claim 1 wherein upon binding of themodulator to the aptamer, the affinity of the aptamer for the targetmolecule is enhanced.
 4. The method according to claim 1 wherein themodulator binds to free aptamer present in the host.
 5. The methodaccording to claim 1 wherein the modulator binds to aptamer ligandpresent in the host in association with the target molecule.
 6. Themethod according to claim 1 wherein the modulator is selected from thegroup consisting of an oligonucleotide complementary to a portion of theaptamer; a nucleic acid binding peptide, polypeptide or protein; a smallmolecule; a ribozyme; a DNAzyme: a peptide nucleic acid (PNA); amorpholino nucleic acid (MNA); a locked nucleic acid (LNA); apseudocyclic oligonucleobase; a molecule that can bind to or otherwisemodulate the activity of the therapeutic nucleic acid ligand; and anucleic acid binding oligosaccharide. 7-51. (canceled)
 52. The methodaccording to claim 6 wherein the modulator is an oligonucleotidecomplementary to a portion of the aptamer.
 53. The method according toclaim 52 wherein the target molecule is selected from the groupconsisting of an intracellular protein, an extracellular protein, and acell surface protein.
 54. The method according to claim 52 wherein thetarget is selected from a receptor, an enzyme, an enzyme inhibitor, ahormone, a glycoprotein and a peptide.
 55. The method according to claim52 wherein the target molecule is selected from the group consisting ofa nicotinic acetylcholine receptor; a platelet receptor gpIIbIIIa; aplatelet receptor gpIbIX; platelet receptor gpVI; a Gas6 receptor; aCTLA4 receptor; a lipid, including a phospholipid, a glycolipid, anucleic acid and a carbohydrate (including a glycosaminoglycan); agrowth factor, a hormone; an interleukin; angiopoietin I; PDGF; VEGF;angiogenesis factor I (AngI); angiogenesis factor 2 (Ang2); thrombin;and an interleukin; an E2F transcription factor; a small molecule suchas glucose; a tissue factor/coagulation factor VIIa enzyme complex(TF/FVIIa); a coagulation factor VIIIa/coagulation factor IXa enzymecomplex (FVIIIa/FIXa); a coagulation factor Va/coagulation factor Xaenzyme complex (FVa/Fxa); plasminogen activator inhibitor 1 (PAI-1);coagulation factor XIIIa (FXIIIa); anti-thrombin III (ATIII); thrombin;and coagulation factor XIa (FXIa).
 56. The method according to claim 6wherein the modulator is a nucleic acid binding peptide, polypeptide orprotein.
 57. The method according to claim 56 wherein the targetmolecule is selected from the group consisting of an intracellularprotein, an extracellular protein, and a cell surface protein.
 58. Themethod according to claim 56 wherein the target is selected from areceptor, an enzyme, an enzyme inhibitor, a hormone, a glycoprotein anda peptide.
 59. The method according to claim 56 wherein the targetmolecule is selected from the group consisting of a nicotinicacetylcholine receptor; a platelet receptor gpIIbIIIa; a plateletreceptor gpIbIX; platelet receptor gpVI; a Gas6 receptor; a CTLA4receptor; a lipid, including a phospholipid, a glycolipid, a nucleicacid and a carbohydrate (including a glycosaminoglycan); a growthfactor, a hormone; an interleukin; angiopoietin I; PDGF; VEGF;angiogenesis factor I (AngI); angiogenesis factor 2 (Ang2); thrombin;and an interleukin; an E2F transcription factor; a small molecule suchas glucose; a tissue factor/coagulation factor VIIa enzyme complex(TF/FVIIa); a coagulation factor VIIIa/coagulation factor IXa enzymecomplex (FVIIIa/FIXa); a coagulation factor Va/coagulation factor Xaenzyme complex (FVa/Fxa); plasminogen activator inhibitor 1 (PAI-1);coagulation factor XIIIa (FXIIIa); anti-thrombin III (ATIII); thrombin;and coagulation factor XIa (FXIa).
 60. The method according to claim 6wherein the modulator is a small molecule.
 61. The method according toclaim 60 wherein the target molecule is selected from the groupconsisting of an intracellular protein, an extracellular protein, and acell surface protein.
 62. The method according to claim 60 wherein thetarget is selected from a receptor, an enzyme, an enzyme inhibitor, ahormone, a glycoprotein and a peptide.
 63. The method according to claim60 wherein the target molecule is selected from the group consisting ofa nicotinic acetylcholine receptor; a platelet receptor gpIIbIIIa; aplatelet receptor gpIbIX; platelet receptor gpVI; a Gas6 receptor; aCTLA4 receptor; a lipid, including a phospholipid, a glycolipid, anucleic acid and a carbohydrate (including a glycosaminoglycan); agrowth factor, a hormone; an interleukin; angiopoietin I; PDGF; VEGF;angiogenesis factor I (AngI); angiogenesis factor 2 (Ang2); thrombin;and an interleukin; an E2F transcription factor; a small molecule suchas glucose; a tissue factor/coagulation factor VIIa enzyme complex(TF/FVIIa); a coagulation factor VIIIa/coagulation factor IXa enzymecomplex (FVIIIa/FIXa); a coagulation factor Va/coagulation factor Xaenzyme complex (FVa/Fxa); plasminogen activator inhibitor 1 (PAI-1);coagulation factor XIIIa (FXIIIa); anti-thrombin III (ATIII); thrombin;and coagulation factor XIa (FXIa).
 64. The method according to claim 6wherein the modulator is a ribozyme or a DNAzyme.
 65. The methodaccording to claim 64 wherein the target molecule is selected from thegroup consisting of an intracellular protein, an extracellular protein,and a cell surface protein.
 66. The method according to claim 64 whereinthe target is selected from a receptor, an enzyme, an enzyme inhibitor,a hormone, a glycoprotein and a peptide.
 67. The method according toclaim 64 wherein the target molecule is selected from the groupconsisting of a nicotinic acetylcholine receptor; a platelet receptorgpIIbIIIa; a platelet receptor gpIbIX; platelet receptor gpVI; a Gas6receptor; a CTLA4 receptor; a lipid, including a phospholipid, aglycolipid, a nucleic acid and a carbohydrate (including aglycosaminoglycan); a growth factor, a hormone; an interleukin;angiopoietin I; PDGF; VEGF; angiogenesis factor I (AngI); angiogenesisfactor 2 (Ang2); thrombin; and an interleukin; an E2F transcriptionfactor; a small molecule such as glucose; a tissue factor/coagulationfactor VIIa enzyme complex (TF/FVIIa); a coagulation factorVIIIa/coagulation factor IXa enzyme complex (FVIIIa/FIXa); a coagulationfactor Va/coagulation factor Xa enzyme complex (FVa/Fxa); plasminogenactivator inhibitor 1 (PAI-1); coagulation factor XIIIa (FXIIIa);anti-thrombin III (ATIII); thrombin; and coagulation factor XIa (FXIa).68. The method according to claim 6 wherein the modulator is a peptidenucleic acid (PNA); a morpholino nucleic acid (MNA) or a locked nucleicacid (LNA).
 69. The method according to claim 68 wherein the targetmolecule is selected from the group consisting of an intracellularprotein, an extracellular protein, and a cell surface protein.
 70. Themethod according to claim 68 wherein the target is selected from areceptor, an enzyme, an enzyme inhibitor, a hormone, a glycoprotein anda peptide.
 71. The method according to claim 68 wherein the targetmolecule is selected from the group consisting of a nicotinicacetylcholine receptor; a platelet receptor gpIIbIIIa; a plateletreceptor gpIbIX; platelet receptor gpVI; a Gas6 receptor; a CTLA4receptor; a lipid, including a phospholipid, a glycolipid, a nucleicacid and a carbohydrate (including a glycosaminoglycan); a growthfactor, a hormone; an interleukin; angiopoietin I; PDGF; VEGF;angiogenesis factor I (AngI); angiogenesis factor 2 (Ang2); thrombin;and an interleukin; an E2F transcription factor; a small molecule suchas glucose; a tissue factor/coagulation factor VIIa enzyme complex(TF/FVIIa); a coagulation factor VIIIa/coagulation factor IXa enzymecomplex (FVIIIa/FIXa); a coagulation factor Va/coagulation factor Xaenzyme complex (FVa/Fxa); plasminogen activator inhibitor 1 (PAI-1);coagulation factor XIIIa (FXIIIa); anti-thrombin II (ATIII); thrombin;and coagulation factor XIa (FXIa).
 72. The method according to claim 6wherein the modulator is a nucleic acid binding oligosaccharide.
 73. Themethod according to claim 72 wherein the target molecule is selectedfrom the group consisting of an intracellular protein, an extracellularprotein, and a cell surface protein.
 74. The method according to claim72 wherein the target is selected from a receptor, an enzyme, an enzymeinhibitor, a hormone, a glycoprotein and a peptide.
 75. The methodaccording to claim 72 wherein the target molecule is selected from thegroup consisting of a nicotinic acetylcholine receptor; a plateletreceptor gpIIbIIIa; a platelet receptor gpIbIX; platelet receptor gpVI;a Gas6 receptor; a CTLA4 receptor; a lipid, including a phospholipid, aglycolipid, a nucleic acid and a carbohydrate (including aglycosaminoglycan); a growth factor, a hormone; an interleukin;angiopoietin I; PDGF; VEGF; angiogenesis factor I (AngI); angiogenesisfactor 2 (Ang2); thrombin; and an interleukin; an E2F transcriptionfactor; a small molecule such as glucose; a tissue factor/coagulationfactor VIIa enzyme complex (TF/FVIIa); a coagulation factorVIIIa/coagulation factor IXa enzyme complex (FVIIIa/FIXa); a coagulationfactor Va/coagulation factor Xa enzyme complex (FVa/Fxa); plasminogenactivator inhibitor 1 (PAI-1); coagulation factor XIIIa (FXIIIa);anti-thrombin III (ATIII); thrombin; and coagulation factor XIa (FXIa).76. The method according to claim 6 wherein the modulator is a moleculethat can bind to or otherwise modulate the activity of the therapeuticnucleic acid ligand.
 77. The method of claim 76 wherein the modulatorchanges the binding of the nucleic acid ligand for its target.
 78. Themethod of claim 76 wherein the modulator degrades, cleaves, metabolizesor breaks down the nucleic acid ligand while the ligand is stillexerting its effect on the target.
 79. The method according to claim 76wherein the target molecule is selected from the group consisting of anintracellular protein, an extracellular protein, and a cell surfaceprotein.
 80. The method according to claim 76 wherein the target isselected from a receptor, an enzyme, an enzyme inhibitor, a hormone, aglycoprotein and a peptide.
 81. The method according to claim 76 whereinthe target molecule is selected from the group consisting of a nicotinicacetylcholine receptor; a platelet receptor gpIIbIIIa; a plateletreceptor gpIbIX; platelet receptor gpVI; a Gas6 receptor; a CTLA4receptor; a lipid, including a phospholipid, a glycolipid, a nucleicacid and a carbohydrate (including a glycosaminoglycan); a growthfactor, a hormone; an interleukin; angiopoietin I; PDGF; VEGF;angiogenesis factor I (AngI); angiogenesis factor 2 (Ang2); thrombin;and an interleukin; an E2F transcription factor; a small molecule suchas glucose; a tissue factor/coagulation factor VIIa enzyme complex(TF/FVIIa); a coagulation factor VIIIa/coagulation factor IXa enzymecomplex (FVIIIa/FIXa); a coagulation factor Va/coagulation factor Xaenzyme complex (FVa/Fxa); plasminogen activator inhibitor 1 (PAI-1);coagulation factor XIIIa (FXIIIa); anti-thrombin III (ATIII); thrombin;and coagulation factor XIa (FXIa).
 82. A method of screening a testcompound for its ability to alter the affinity of an aptamer for atarget molecule comprising: a. contacting the aptamer with the targetmolecule in the presence and absence of the test compound underconditions favoring binding of the aptamer to the target molecule; andb. determining whether the test compound enhances or inhibits binding ofthe aptamer to the target molecule.
 83. A method of altering theaffinity of an aptamer for a target molecule in vitro comprisingcontacting the aptamer with a modulator in vitro under conditions suchthat the modulator binds to the aptamer and thereby alters the affinityof the aptamer for the target molecule, wherein the target molecule isnot thrombin.